Amino acids and processes for making peptides using same

ABSTRACT

Methods of making unnatural amino acids are provided which unnatural amino acids can be incorporated into peptides which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. These unnatural amino acids are useful in the synthesis of peptides and have the formula (a): ##STR1## where W is (CH 2 ) or ##STR2## n is an integer from 1 to 6; j=1, 2 or 3, and preferably, Y is N--CN, X is NH and R 2  is alkyl, modified alkyl, alkenyl, alkynyl, aryl or methyl pyridyl. Disclosed are peptides that are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions.

This invention was made with Government support under grant numberHD-13527 and contracts NO1-HD-1-3100 and NO1-HD-0-2906 awarded by theNational Institutes of Health. The Government has certain rights in thisinvention.

This application is a divisional of U.S. Ser. No. 08/289,103, filed Aug.11, 1994, now U.S. Pat. No. 5,565,574, which is a divisional of U.S.Ser. No. 08/078,965, filed Jun. 17, 1993, now U.S. Pat. No. 5,352,796,which is a continuation-in-part of U.S. Ser. No. 08/006,729, filed Jan.21, 1993, now U.S. Pat. No. 5,296,468, which is a continuation-in-partof U.S. Ser. No. 07/669,695, filed Mar. 14, 1991, now abandoned, whichis a continuation-in-part of U.S. Ser. No. 07/545,239, filed Jun. 27,1990, now U.S. Pat. No. 5,169,932, which is a continuation-in-part ofU.S. Ser. No. 07/428,827, filed Oct. 30, 1989, now abandoned.

This invention relates generally to unnatural amino acids and to thepreparation of new unnatural amino acids, which may be derived fromdiamino acids, such as Lys, Orn, Dpr, Dbu and particularly 4NH₂ Phe,which amino acids are useful in the synthesis of biologically activepeptides. More particularly, it relates to peptides having suchunnatural amino acids which are prepared either by modifying commonlyavailable amino acids in a fully assembled peptide or by forming theprotected unnatural amino acid and then incorporating it into such apeptide as a part of a standard chain elongation synthesis process.

In one more particular aspect, the present invention provides GnRHantagonists which inhibit gonadal function and the release of thesteroidal hormones, progesterone and testosterone and also to peptideswhich promote the release of such steroids.

BACKGROUND OF THE INVENTION

The pituitary gland is attached by a stalk to the region in the base ofthe brain known as the hypothalamus. In particular, follicle stimulatinghormone (FSH) and luteinizing hormone (LH), sometimes referred to asgonadotropins or gonadotropic hormones, are released by the pituitarygland. These hormones, in combination, regulate the functioning of thegonads to produce testosterone in the testes and progesterone andestrogen in the ovaries, and they also regulate the production andmaturation of gametes.

The release of a hormone by the anterior lobe of the pituitary glandusually requires a prior release of another class of hormones producedby the hypothalamus. One of the hypothalamic hormones acts as a factorthat triggers the release of the gonadotropic hormones, particularly LH,and this hormone is referred to herein as GnRH although it has also beenreferred to as LH--RH and as LRF. GnRH was isolated and characterized asa decapeptide some 20 years ago, and it was found that analogs of GnRHhaving a D-isomer instead of Gly in the 6-position, such as D-Ala⁶!-GnRH (U.S. Pat. No. 4,072,668) having the following formula:

pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH₂, have greater bindingstrength to the receptor and greater biological potency than the nativehormone both in vitro and in vivo.

Peptides are compounds which contain two or more amino acids in whichthe carboxyl group of one acid is linked to the amino group of the otheracid. The formula for GnRH, as represented above, is in accordance withconventional representation of peptides where the amino terminus appearsto the left and the carboxyl terminus to the right. The position of theamino acid residue is identified by numbering the amino acid residuesfrom left to right. In the case of GnRH, the hydroxyl portion of thecarboxyl group of glycine at the C-terminus has been replaced with anamino group(NH₂) i.e. the C-terminus is amidated. The abbreviations forthe individual amino acid residues above are conventional and are basedon the trivial name of the amino acid, e.g. pGlu is pyroglutamic acid,Glu is glutamic acid, His is histidine, Trp is tryptophan, Ser isserine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Nle isnorleucine, Orn is ornithine, Arg is arginine, Har is homoarginine, Prois proline, Sar is sarcosine, Phe is phenylalanine, Ala is alanine, Valis valine, Nva is norvaline, Ile is isoleucine, Thr is threonine, Lys islysine, Asp is aspartic acid, Ash is asparagine, Gln is glutamine, andMet is methionine. Except for glycine, amino acids of the peptides ofthe invention are of the L-configuration unless noted otherwise.

There are reasons for desiring to prevent ovulation in femalemammalians, and the administration of GnRH analogs that are antagonisticto the normal function of GnRH have been used to suppress or delayovulation. For this reason, analogs of GnRH which are antagonistic toGnRH are being investigated for their potential use as a contraceptiveor for regulating conception periods. GnRH antagonists may also be usedfor the treatment of precocious puberty and endometriosis. Suchantagonists have also been found useful to regulate the secretion ofgonadotropins in male mammals and can be employed to arrestspermatogenesis, e.g. as male contraceptives for treatment of male sexoffenders, and for treatment of prostatic hypertrophy. Morespecifically, GnRH antagonists can be used to treat steroid-dependenttumors, such as prostatic and mammary tumors, and for the control of thetiming of ovulation for in vitro fertilization. In the female, they canalso be used to treat hirsutism, endometriosis, premenstrual syndrome(PMS), etc.

On the other hand, GnRH agonists function in the same manner as GnRH inpromoting the release of LH and FSH, and agonists which exhibit greaterbiopotency and/or longer duration of action are considered valuable.

In one aspect, it is desired to provide improved peptides which eitherare strongly antagonistic to endogenous GnRH and prevent secretion of LHand FSH and the release of steroids by the gonads of mammals or arestrong agonists of GnRH. Of particular interest are compounds which aremore effective in vivo when administered orally. As indicatedhereinafter, 6-position modifications in the form of D-isomers are knownto improve biological activity, and improved modifications to thenatural amino acids have been sought to further enhance biologicalactivity as a result of their substitution into this and other positionsin these peptide analogs.

SUMMARY OF THE INVENTION

The present invention provides unnatural amino acids and methods formaking same, as well as peptides that can be prepared either using suchpreviously prepared unnatural amino acids or by modifying a previouslyprepared peptide (or protected peptide-resin) containing a desiredoverall sequence which includes one or more amino acid residues havingan amino group which is to be modified. Preferred amino acids of theinvention have a side chain which contains a modified guanidino group ora guanidino equivalent or a derivative that is obtained by furtherelucidation of a modified guanidino group, as set forth hereinafter, andwhich most preferably contains a triazole or substituted triazole group.

In another particular aspect, the invention provides peptides whichinhibit the release of gonadotropins in mammalians, including humans,and it also provides methods for inhibiting the release of steroids bythe gonads of male and female mammalians. The invention also providesimproved GnRH analogs which are strong agonists of GnRH and can be usedto promote the reproduction processes of mammalians. As mentioned above,these GnRH antagonists may be used to inhibit the production ofgonadotropins and sex hormones under various circumstances, includingprecocious puberty, hormone dependent neoplasia, dysmenorrhea,endometriosis, steroid-dependent tumors and PMS.

The invention provides unnatural amino acids having the followingformula U*: ##STR3## where W is (CH₂)_(n) or ##STR4## n is an integerfrom 1 to 6 and is preferably 1, 2, 3 or 4; j is 1, 2 or 3; Y=N--CN,N--CONHR₉, O, S or CH--NO₂, where R₉ is H, Ac, alkyl (preferably C₁ toC₄), naphthyl, pyridyl, pyrimidyl, pyrazinyl, indolyl, quinolinyl orimidazolyl, which alkyl and cyclic groups are unsubstituted orsubstituted (preferably by chloro, fluoro, bromo, amino, nitro, alkyl(C₁ to C₄) and alkoxy (C₁ to C₄)); X═NH, S, O, N₃, M₁ --(CH_(q))_(p)--M₂ or M₁ --(CH₂)_(p') --M₂ (CH₂)_(p") --M₃, wherein M₁ is NR₁₀, S, Oor CHR₃ wherein R₃ is methyl, ethyl, propyl, phenyl, pyridinyl,pyrimidinyl or purinyl; q=1 or 2; p, p' and p" are integers between 0and 6; R₁₀ is H, methyl, ethyl, propyl, phenyl or substituted phenyl(preferably by Cl, F, NO₂ or NH₂) or an acyl radical having from 1 to 6carbons, such as acetyl; and M₂ and M₃ ═M₁, COOH, CONH₂, COOR₃ or CN(preferably X is NH or O); R₁ ═H, alkyl (preferably C₁ to C₆ and mostpreferably C₁ to C₄), modified alkyl (preferably C₁ to C₅, the terminalcarbon of which is either substituted with NH₂, OH, Cl, Br or F or isreplaced with CF₃ or CF₂ CF₃), alkenyl (preferably C₂ to C₄), such asCH₂ CH═CHR₃, alkynyl (preferably C₂ to C₄), such as CH₂ C═CR₃, aryl suchas benzyl, tolyl, p-amino-benzyl (anilinyl) and pCl-benzyl or a directbond to X; R₂ ═R₁, OH, NH₂, NHR₁, heterocycle (preferably as illustratedhereinafter) or desR₂, with R₂ being desR₂ when X═N₃. Optionally R₂ andX can be interconnected, or R₁ and R₂ can be connected to each other viaa branched or unbranched methylene bridge of type --(CH₂)_(m) -- or--(CH₂)_(m) --M--(CH₂)_(m') --. In such an R₁ -R₂ moiety, m and m' areintegers from 1 to 6 and preferably from 1 to 3; and M═NH, O, S or CHR₄,wherein R₄ is lower alkyl or aryl and is preferably methyl, ethyl,propyl, phenyl or pCl-phenyl, with M preferably being O or S. Mostpreferably, when R₁ and R₂ are interconnected, they form a 5, 6, or7-member heterocyclic ring with the "N--C--X" portion of the formula U*.If desired to form a cyclic peptide, XR₂ can contain a part of anotherdiamino acid within the same peptide, e.g., the omega amino group of the5-position residue can be so linked to such an unnatural amino acidresidue in the 8-position.

Preferably the unnatural amino acid has one of the following formulas:##STR5## where W is (CH₂)_(n) or ##STR6## n is an integer from 1 to 6; jis 1, 2 or 3; Y is N--CN, N--CONHR₉, S, O or CH--NO₂ where R₉ is H, Ac,alkyl, naphthyl, pyridyl, pyrimidyl, pyrazinyl, indolyl, quinolinyl orimidazolyl; X is NH, O, S, N₃, M₁ (CH_(q))_(p) M₂ or M₁ --(CH₂)_(p')--M₂ (CH₂)_(p") --M₃, where M₁ is NR₁₀, N, O, S or CHR₃ wherein R₃ ismethyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl; q is 1or 2; p and p" are integers between 1 and 6; p' is an integer from 0 to6; R₁₀ is H, methyl, ethyl, propyl, phenyl or phenyl substituted by Cl,F, NO₂ or NH₂ ; M₂ and M₃ are M₁, COOH, CONH₂, COOR₃ or CN; R₁ ═H, alkyl(C₁ to C₆), (CH₂)_(n') X", alkenyl (C₂ to C₄), alkynyl (C₂ to C₄), oraryl; R₂ is H, alkyl (C₁ to C₆), (CH₂)_(n') X", alkenyl (C₂ to C₄),alkynyl (C₂ to C₄), aryl, OH, NH₂, NHR, heterocycle or des R₂, with R₂being desR₂ when X is N₃ ; R is lower alkyl; n' is 1, 2, 3 or 4; and X"is CH₂ NH₂, CH₂ OH, CH₂ Cl, CH₂ Br, CH₂ F, CF₃ or CF₂ CF₃ ; ##STR7##where W is (CH₂)_(n) or ##STR8## n is an integer from 1 to 6; j is 1, 2or 3; M₁ is NR₁₀, O, S or CHR₃ ; R₃ is methyl, ethyl, propyl, phenyl,pyridinyl, pyrimidinyl or purinyl; R₁₀ is H, methyl, ethyl, propyl,phenyl on phenyl substituted by Cl, F, NO₂ or NH₂ and R₁₁ is H oracyl(C₁ to C₆); ##STR9## where W is (CH₂)_(n) or ##STR10## n is aninteger from 1 to 6; j is 1, 2 or 3; M₁ is NR₁₀, O, S or CHR₃ wherein R₃is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl; R₁₀is H, methyl, ethyl, propyl, phenyl or phenyl substituted by Cl, F, NO₂or NH₂ ; R₂ is H, alkyl (C₁ to C₆), (CH₂)_(n) X", alkenyl (C₂ to C₄),alkynyl (C₂ to C₄), aryl, OH, NH₂, NHR, or heterocycle; R is loweralkyl; n' is 1, 2, 3 or 4; and X" is CH₂ NH₂, CH₂ OH, CH₂ Cl, CH₂ Br,CH₂ F, CF₃ or CF₂ CF₃ ; ##STR11## where W is (CH₂)_(n) or ##STR12## n isan integer from 1 to 6; j is 1, 2 or 3; Y is N--CN, S, or O; M₁ is NR₁₀,or CHR₃ ; R₃ is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl orpurinyl; p is an integer between 1 and 6; and R₁₀ is H, methyl, ethyl,propyl, phenyl or phenyl substituted by Cl, F, NO₂ or NH₂ ; or ##STR13##where W is (CH₂)_(n) or ##STR14## n is an integer from 1 to 6; j is 1, 2or 3; Y is N--CN, N--CONH₉, S, O or CH--NO₂ ; R₉ is H, Ac, alkyl,naphthyl, pyridyl, pyrimidyl, pyrazinyl, indolyl, quinolinyl orimidazolyl; X is NH, O or S; and R₁ -R₂ is either --(CH₂)_(m) -- or--(CH₂)_(m) --M--(CH₂)_(m) -- where m and m' are integers from 1 to 6and M is NH, O, S or CHR₄, with R₄ being lower alkyl or aryl.

More preferably, the amino acid has the formula: ##STR15## where W is aspreviously defined.

Most preferably, W in the foregoing formula is ##STR16## Such aminoacids are valuable in both L- and D-isomer forms to synthesize GnRHanalogs and a wide variety of other peptides by the standard chainelongation methods.

Modification of the specified primary amino function of a natural orinitially modified α-amino acid (or a peptide containing same) iscarried out by treatment of either the appropriately protected peptideor amino acid with an appropriate reagent(s). Peptides or amino acidswhere Y is N--CN (herein referred to as cyanoguanidines) are prepared byreaction of an amino group with diphenyl cyanocarbonimidate (I):##STR17## wherein "Q" is used to broadly represent either the majorportion of a peptide or an amino acid having a primary amino group (suchas the amino acid which is depicted above) as a part of formula U*.

The peptide or amino acid having theN-substituted-N'-cyano-O-phenylisourea moiety (II) can then be eitherisolated or further functionalized by reaction with a second nucleophileHXR₂ to produce cyanoguanidine-containing peptides or amino acids havingthe formula (III): ##STR18## For example, ##STR19## For example, whereHXR₂ ═H₂ N--CH₂ --pyridine, the result is: ##STR20## This group may alsobe referred to (IUPAC nomenclature) asN-g-cyano-N-g'-3-methylpyridylguanidino.

Such compounds can be hydrolyzed under acidic conditions to producecompounds which are also biopotent--for example: ##STR21## Thehydrolyzed versions, referred to herein as including the N-g'-amidogroup, can also be synthesized directly by reacting phosgene derivateswith moieties having a guanidino function.

If HXR₂ is an amino group of another peptide or protein, one will obtaina peptide-peptide dimer or peptide-protein dimer conjugated via thecyanoguanidine moiety. If HXR₂ is the N-terminal primary amino group orthe side chain amino group of another amino acid in the same peptide,one will obtain a cyclic peptide (IV) linked via the cyanoguanidinemoiety: ##STR22## wherein Q₁ and Q₂ represent the remainders of twoamino acid residues in the same peptide. Cyclization via thecyanoguanidine derivative is preferably effected while a part of thepeptidoresin, as opposed to subsequently cyclizing the linear peptide.

A special case arises when --XR₂ contains a second nucleophilic site andX has the general form: M₁ --(CH_(q))_(p) --M₂ or M₁ --(CH₂)_(p') --M₂--(CH₂)_(p") --M₃, where M₁, M₂ and M₃ are individually NH, N, O orCHR₃, with p, p', p" being 0, 1, 2 or 3 and q being 1 or 2. Examples ofsuch nucleophiles include H₂ NNH₂, CH₃ HNNH₂, CH₃ HNNHCH₃, H₂ NOH, andH₂ N--CH₂ --CH₂ OH. In this case, the cyanoguanidine moiety that isformed can be converted into the corresponding heterocycle (V) whichforms from the initial intermediate by reaction of the omega amino groupwith the cyano group such as: ##STR23## For example, where --XR₂═--HNNH₂, ##STR24## which may then undergo hydrolysis, as indicatedabove.

Where XR₂ contains a carboxylic acid group or the equivalent,particularly a carboxylic ester or carboxylic amide, a heterocyclicmoiety, such as a saturated pyrimidine-like moiety (VI), is formed, byreaction of the carboxylic group with the secondary amino group (R₁),when M₁ is N, and similar 6-membered heterocyclic moieties are formedwhen M₁ is O or S. For example, R₂ may be M₁ --(CH₂)_(p) --M₂ with M₂═COOH, COOCH₃ or CONH₂ and p being an integer between 1 and 4. Forinstance in such a case where an aliphatic carboxylic acid group ispresent and p=2: ##STR25## If R₂ includes an ortho-substituted aromaticcarboxylic acid, e.g. benzoic acid (q=1 and p=6), the correspondingquinazoline-like species (VII) is formed: ##STR26## Such benzoic acidmay be further substituted, and such substitutions may in any of theother 4 ring positions, as shown, creating the corresponding substitutedquinazoline-like moiety which is considered to be equivalent to theunsubstituted. X' may be H, Cl, Br, F, NHCH₃ or SCH₃, and R₇ and R₈ maybe H, CH₃ or CH₂ CH₃.

The molecules wherein X═N₃ and R₂ is desR₂ (i.e. deleted) are useful forphotolabeling because of the activity of the --N₃ group and are formedby reacting the moiety (II) with sodium azide (NAN₃).

Peptides wherein Y is O (herein referred to as ureas) or S (referred toas thioureas) are prepared by the well known procedure in which thedesired side chain amino group is treated with an appropriate isocyanateor thioisocyanate to obtain such ureas or thioureas. ##STR27##

Peptides or amino acids wherein Y is CH--NO₂ (herein referred to asdiaminonitroethylenes) are prepared by conversion of the correspondingurea to a carbodiimide: ##STR28## followed by treatment withnitromethane anion (prepared by the action of sodium hydride onnitromethane in dry DMF) as disclosed generally in F. Meimas, et al.,Synthesis, 509-510 (1985): ##STR29##

An alternative synthesis that may be used is as follows: ##STR30##

Generally, in accordance with the present invention, peptides aresynthesized which are antagonists or agonists of GnRH, i.e., they eitherstrongly inhibit the secretion of gonadotropins by the pituitary glandof mammalians, including humans, and/or inhibit the release of steroidsby the gonads, or they strongly promote such secretion or release. Thesepeptides are analogs of GnRH containing one or more unnatural aminoacids of the formula U* in the 3-position, the 5-position, the6-position and/or the 8-position. When U* is in the 3-and/or 6-position,it is always in the form of a D-isomer; whereas when U* is in the 5-and/or 8-position, it is always in the form of an L-isomer. Anantagonist should have a 1-position substitution, such as dehydroPro orβ-(1-or 2-naphthyl)-D-alanine (hereinafter β-D-1NAL or β-D-2NAL), a2-position substitution in the form of a modified D-Phe and a 3-positionsubstitution, preferably in the form of substituted or unsubstitutedD-Trp, D-3PAL, β-D-NAL or the residue of a D-isomer amino acid U*. The5-position may be occupied by (a) Tyr, (b) a halogenated or methylatedPhe or Tyr, (c) Arg, (d) Lys in which the side chain amino group isacylated by 3-carboxypyridine (nicotinic acid) or by 2 or4-carboxypyridine, i.e. Lys(cpd), preferably Lys(3cpd) which is alsoreferred to as Lys(Nic), (e) His or (f) the residue of an L-isomer aminoacid U*. Agonists have a 6-position substitution which is the residue ofthe D-isomer U*, and the antagonists have either a D-isomer U* or asubstituted or acylated D-Lys in the 6-position. Instead of Leu in the7-position, both may have Nle, NML, Phe, Nva, Met, Tyr, Trp or PAL, ofwhich the Phe or Trp may be substituted. The antagonists may also havean optional substitution in the 8-position, which preferably may be theL-isomer U* or isopropyl Lys, i.e., ILys or Lys(Ipr) wherein the sidechain amino group is substituted by isopropyl, and a substitution in the10-position such as D-Ala. At least one residue of a D-isomer amino acidof the formula U* is most preferably present in each peptide of theinvention.

Modified D-Phe in the 2-position provides increased antagonisticactivity as a result of the specific modifications present in thebenzene ring. Single substitutions for hydrogen in the ring arepreferably made in the para- or 4-position, but might be in either the2- or 3-position also; the substitutions are selected from chloro,fluoro, bromo, methyl, methoxy and nitro, with chloro, fluoro and nitrobeing preferred. Dichloro substitutions are in the 2,4 or 3,4 positionsin the ring. The α-carbon atom may also be methylated, e.g. (C.sup.αMe/4Cl)Phe. The 1-position substituent is preferably modified so thatits α-amino group contains an acyl group, such as formyl(For),acetyl(Ac), acrylyl(Acr), vinylacetyl(Vac) or benzoyl(Bz), with acetyland acrylyl being preferred and with acetyl being most preferred. PALand D-PAL represent the L- and D-isomers of pyridylalanine where theβ-carbon of Ala is linked to the 2-, 3- or 4-position, preferably to the3-position, on the pyridine ring. When β-D-NAL is present in the1-position and R₅ is not Arg, a hydrophilic D-amino acid residue, suchas 4NH₂ -D-Phe, 4-guanidino-D-Phe, D-His, D-Lys, D-Orn, D-Arg,D-Har(Homoarginine) or D-PAL is preferably present in the 6-position ifU* is not present. When dehydroPro is present in the 1-position, D-PALor a D-isomer of a lipophilic amino acid, such as D-Trp, D-Phe,For-D-Trp, NO₂ -D-Trp, D-Leu, D-Ile, D-Nle, D-Tyr, D-Val, D-Ala, dialkylArg, dialkyl Har, D-Ser(OtBu), β-D-NAL or (imBzl)D-His is preferably inthe 6-position, if U* is not present.

These GnRH analogs are very soluble at a pH just below physiological pH,i.e. about 4.5 to about 6, and thus can be formulated and administeredin concentrated form, greatly facilitating administration at a pH ofabout 5 to 7.4 which is presently preferred. The antagonists inhibitovulation of female mammals when administered at low levels at proestrusand are also effective to cause resorption of fertilized eggs ifadministered shortly after conception. The antagonists are alsoeffective for the contraceptive treatment of male mammals and thetreatment of steroid-dependent tumors. Certain of the antagonists aresurprisingly long-acting in their suppression of LH levels followingadministration, and certain have a particularly low side effect inrespect of histamine release. The agonists are substantially more potentthan native GnRH in effecting release of LH and FSH.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As previously mentioned, the unnatural amino acids (which can be L- orD-isomers) are represented by the formula U* ##STR31## wherein W, X, Y,R₁ and R₂ are as defined previously, and the α-amino group may besubstituted by a suitable protecting group, e.g. Boc, to permit its useas a part of a chain elongation method for synthesizing a peptidebeginning at the C-terminus. In each peptide of the invention, there isat least one such residue (preferably a D-isomer).

More specifically, the GnRH antagonists of the present invention arerepresented by the following Formula (F₁):

G-AA₁ -(A)D-Phe-AA₃ -Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein G ishydrogen or an acyl group having 7 or less carbon atoms; AA₁ isdehydroPro, D-pGlu, (A)D-Phe, (B)D-Trp, Pro, or β-D-NAL; A is H, Cl, F,NO₂, CH₃, OCH₃, C.sup.α Me/4Cl, Cl₂ or Br; B is H, NO₂, NH₂, OCH₃, F,Cl, Br, CH₃, N^(in) For or N^(in) Ac; AA₃ is U*, D-PAL, β-D-NAL or(B)D-Trp; AA₅ is U*, Tyr, (C)Arg, Lys(cpd), Orn(cpd), Dbu(cpd),Dpr(cpd), (A)Phe, (3I)Tyr or His; AA₆ is U*, β-D-NAL, (B)D-Trp,(A')D-Phe, (D)D-Orn, (D)D-Lys, (D)D-Dbu, (D)D-Dpr, D-Har, D-Tyr,(E)D-His, D-PAL, (C)D-Arg or a suitable lipophilic D-isomer; A' is A,NH₂, NHCH₃ or gua; C is H or lower alkyl; D is G, cpd or an aryl group;E is H, imBzl or dinitrophenol; AA₇ is Nle, Leu, NML, (A)Phe, Met, Nva,Tyr, (B)Trp or PAL; AA₈ is U*, (C')Arg, (C')Har or ILys; C' is H ordi-lower alkyl; AA₁₀ is D-Ala-NH₂, Gly-NH₂, AzaGly-NH₂ or NH(R); R islower alkyl, preferably CH₂ CH₃ ; and U* is as defined above. When AA₁is β-D-NAL and AA₅ is not Arg, then AA₆ is preferably U*, 4-NH₂ -D-Phe,D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D-Arg.

By dehydroPro is meant 3,4 dehydroproline, C₅ H₇ O₂ N. By β-D-NAL ismeant the D-isomer of alanine which is substituted by naphthyl on theβ-carbon atom, i.e., also 3-D-NAL. Preferably β-D-2NAL is employedwherein the attachment to naphthalene is at the 2-position on the ringstructure; however, β-D-1NAL may also be used. The preferred 1-positionresidues are β-D-NAL, substituted D-Phe and optionally substitutedD-Trp. PAL represents alanine which is substituted by pyridyl on theβ-carbon atom; preferably the linkage is to the 3-position on thepyridine ring. When substituted D-Trp is employed, single substitutionsfor hydrogen are preferably made in either the 5- or 6-position, whichare selected from chloro, fluoro, bromo, methyl, amino, methoxy andnitro, with chloro, fluoro and nitro being preferred. Alternatively, theindole nitrogen may be acylated, e.g. with formyl (N^(in) For- or 1For-)or with acetyl. D-3PAL, N^(in) For-D-Trp and 6NO₂ -D-Trp are thepreferred residues for the 3-position although D-Trp is also often used.When U* is not in the 5-position, it is preferably Tyr, Arg or Lys(cpd).By NML is meant NaCH₃ -L-Leu. By Dbu is meant alpha, gamma diaminobutyric acid, and by Dpr is meant α,β diamino propionic acid. By Aph ismeant 4NH₂ Phe. When dehydroPro is present in the 1-position, Tyr or U*is preferably present in the 5-position and a lipophilic residue is inthe 6-position. By 4-gua-D-Phe is meant a residue of D-Phe havingguanidine substituted in the para-position. By AzaGly-NH₂ is meantNHNHCONH₂. The guanidino group of an Arg residue in the 5- or 6-positionmay be substituted by lower alkyl, i.e. 1 to 4 carbon atoms, e.g.,propyl(Pr). When D-Lys, D-Dbu, D-Dpr or D-Orn is present in the6-position and it is not a part of an unusual amino acid U*, itsside-chain-amino group may be acylated by an acyl group which may bealiphatic, heterocyclic or aromatic, e.g. nicotinic acid, or may besubstituted by an aryl group having not more than 1 phenyl ring. When U*is not present in the 6-position, it is preferably D-PAL or D-Lys(cpd).The 7-position residue is preferably Leu, NML, Nle or Phe. If the8-position residue is not U*, it is preferably ILys.

More specifically the GnRH agonists of the invention are represented bythe following Formula (F₂):

pGlu-His-Trp-Ser-Tyr-U*-AA₇ -Arg-Pro-AA₁₀, wherein AA₇ and AA₁₀ are asdefined hereinbefore; preferably AA₇ is Leu or NML and AA₁₀ is NHCH₂CH₃.

Overall, the invention thus provides GnRH analogs having the Formula(F₃):

G-AA-AA₂ -AA'-Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein AA is pGlu or AA₁; AA₂ is His or (A)D-Phe; AA' is Trp or AA₃ ; and all others are asdefined hereinbefore.

One preferred subgenus of GnRH antagonists has the following formula:

Ac-AA₁ -(A) D-Phe-AA₃ -Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein AA₁ is(A)D-Phe, (B)D-Trp or β-D-NAL; A is H, 4Cl, 4F, 4NO₂, 4CH₃, 4OCH₃,C.sup.α Me/4Cl, 2,4Cl₂ or 4Br; B is H, 6NO₂, 6NH₂, 6OCH₃, 6F, 6Cl, 6Br,6CH₃, N^(in) For or N^(in) Ac; AA₃ is U*, D-PAL, β-D-NAL or (B)D-Trp;AA₅ is U*, Lys(cpd) or Tyr; AA₆ is U*, β-D-NAL, 4NH₂ D-Phe, (B)D-Trp,D-Lys(cpd), D-PAL or D-Arg; AA₇ is Nle, Leu, NML or Phe; AA₈ is U*,ILys, or Arg; AA₁₀ is D-Ala-NH₂, Gly-NH₂, NHNHCONH₂ or NH(R); R is loweralkyl; and U* is either ##STR32## where W is (CH₂)_(n) or ##STR33## n isan integer from 1 to 4; j is 1,2 or 3; X is NH or O; Y is N--CN orN--CONHR₉ where R₉ is H or lower alkyl; R₂ is lower alkyl, cyclohexyl,phenyl, pyridyl, methyl pyridyl or histaminyl; or ##STR34## where W isdefined as above and R₁₁ is H or an acyl radical having 1 to 6 carbonatoms; provided, however, that at least one of AA₃, AA₅, AA₆ and AA₈ isU*.

Another preferred subgenus of GnRH antagonists has the followingformula:

Ac-AA₁ -(A)D-Phe-U*-Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein AA₁ is(A)D-Phe, (B)D-Trp or β-D-NAL; A is H, 4Cl,4F, 4NO₂, 4CH₃, 4OCH₃,C.sup.α Me/4Cl, 2,4Cl₂ or 4Br; B is H, 6NO₂, 6NH₂, 6OCH₃, 6F, 6Cl, 6Br,6CH₃, N^(in) For or N^(in) Ac; AA₅ is Lys(cpd) or Tyr; AA₆ is β-D-NAL,4NH₂ D-Phe, (B)D-Trp, D-Lys(cpd), D-PAL or D-Arg; AA₇ is Nle, Leu, NMLor Phe; AA₈ is ILys or Arg; AA₁₀ is D-Ala-NH₂, Gly-NH₂, NHNHCONH₂ orNH(R); R is lower alkyl; and U* is either ##STR35## where W is (CH₂)_(n)or ##STR36## n is an integer from 1 to 4; j is 1,2 or 3 (preferably 1);X is NH or O; Y is N--CN or N--CONH₉ where R₉ is H or lower alkyl (C₁-C₃), preferably H; R₂ is lower alkyl (C₁ -C₆), cyclohexyl, phenyl,pyridyl, methyl pyridyl or histaminyl; or ##STR37## where W is definedas above and R₁₁ is H or an acyl radical having 1 to 3 carbon atoms,preferably acetyl.

Still another preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-U*-Ser-Tyr-D-3PAL-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂,wherein U* is a D-isomer having the formula either ##STR38## where R₂ islower alkyl.

Yet another preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-U*-U*-AA₇ -Lys(isopropyl)-Pro-AA₁₀wherein AA₇ is Leu or N.sup.α CH₃ Leu; AA₁₀ is D-Ala-NH₂, Gly-NH₂, orNHCH₂ CH₃ ; and U* is either ##STR39## where R₂ is lower alkyl, pyridylor methyl pyridyl;

provided, however, that U* in the 6-position is always a D-isomer.

An additional preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL- (4Cl)D-Phe-D-3PAL-Ser-U*-U*-AA₇ -Lys(isopropyl)-Pro-AA₁₀wherein AA₇ is Leu or N.sup.α CH₃ -Leu; AA₁₀ is D-Ala-NH₂, Gly-NH₂ orNHCH₂ CH₃ ; and U* is ##STR40## where W is (CH₂)_(n) or ##STR41## n isan integer from 1 to 6; and R₁₁ is H or an acyl radical having 1 to 6carbon atoms; provided, however, U* in the 6-position is a D-isomer.

One more preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂ wherein AA₅ is U* orLys(carboxypyridine); AA₆ is U* or D-Lys(carboxypyridine); and U* is##STR42## where W is (CH₂)_(n) and n is 4; provided, however, thateither AA₅ is U* or AA₆ is U*, with AA₆ always being a D-isomer.

A preferred subgenus of amino acids has the formula: ##STR43## where jinteger from 1 to 3; Y is N--CN; X is NH; R₂ is H, alkyl, (CH₂)_(n) X",alkenyl alkynyl aryl OH, NH₂ or heterocycle such as methyl pyridyl,pyrimidinyl or purinyl; wherein n' is 1, 2, 3 or 4, and X" is CH₂ NH₂,CH₂ OH, CH₂ Cl, CH₂ Br, CH₂ F, CF₃ or CF₂ CF₃.

Another preferred subgenus of amino acids are those having the formula:##STR44## where W is (CH₂)_(n) or ##STR45## n is an integer from 1 to 6;and R₁₁ is H or an acyl radical having 1 to 6 carbon atoms. Preferably,W is ##STR46## and R₁₁ is H or acetyl; most preferably, R₁₁ is H.

The peptides of the present invention can be synthesized by classicalsolution synthesis, but are preferably synthesized by a solid phasetechnique. A chloromethylated resin or a hydroxymethylated resin may beused; however, a methylbenzhydrylamine(MBHA) resin, a benzhydrylamine(BHA) resin or some other suitable resin known in the art which directlyprovides a C-terminal amide or substituted amide upon cleavage ispreferably employed when such a C-terminus is desired. For example,peptides having a substituted amide at the C-terminus are preferablysynthesized using an N-alkylamino methyl resin as taught in U.S. Pat.No. 4,569,967, issued Feb. 11, 1986. Solid phase synthesis is conductedin a manner to stepwise add amino acids in the chain in the manner setforth in detail in the U.S. Pat. No. 4,211,693. Side-chain protectinggroups, as are well known in the art, are preferably included as a partof any amino acid which has a particularly reactive side chain andoptionally in the case of others, such as Trp, which amino acids are tobe coupled in the chain being built upon the resin. Such synthesisprovides the fully protected intermediate peptidoresin.

Chemical intermediates made by solution or solid-phase synthesis may berepresented by the formula: X¹ -AA-AA₂ (X⁵)-U₃ -Ser (X³)-U₅ -U₆ -AA₇ (X²or X⁷)-U₈ -Pro-X⁸ wherein: U₃ is either U' or AA'(X²); U₅ is either U'or AA₅ (X⁴ or X⁵); U₆ is either U' or AA₆ (X⁴ or X⁵ or X⁶); U₈ is eitherU' or AA₈ (X⁵ or X⁶); U' is either Lys(X^(a)), Aph(X^(a)), Orn(X^(a)),Dbu(X^(a)) or Dpr(X^(a)); X¹ is an α-amino protecting group of the typeknown to be useful in the art in the stepwise synthesis of polypeptidesand when G in the desired peptide composition is a particular acylgroup, that group may be used as the protecting group. Among the classesof α-amino protecting groups covered by X¹ are (1) acyl-type protectinggroups, such as formyl(For), trifluoroacetyl, phthalyl,p-toluenesulfonyl(Tos), benzoyl(Bz), benzenesulfonyl,dithiasuccinoyl(Dts), o-nitrophenylsulfenyl(Nps), tritylsulfenyl,o-nitrophenoxyacetyl, acrylyl(Acr), chloroacetyl, acetyl(Ac) andγ-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g.,benzyloxycarbonyl(Z), fluorenylmethyloxycarbonyl(Fmoc) and substitutedbenzyloxycarbonyl, such as p-chlorobenzyloxy-carbonyl(ClZ),p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl andp-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups,such as tertbutyloxycarbonyl(Boc), diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4)cycloalkyl urethan-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl;(5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6)alkyl-type protecting groups, such as allyl(Aly),triphenylmethyl(trityl) and benzyl(Bzl); (7) trialkylsilane groups, suchas trimethylsilane. The preferred α-amino protecting group is Boc when Xis hydrogen.

X² is hydrogen or a protecting group for the indole nitrogen of Trp,such as Bz, Ac or For. In many syntheses there is no need to protectTrp, and such protection is not used if acylated D-Trp is presentelsewhere in the peptide.

X³ is a protecting group for the hydroxyl side chain of Ser or Thr, e.g.Ac, Bz, trityl, DCB or benzyl ether(Bzl) and is preferably Bzl.

X⁴ is hydrogen or a protecting group for the phenolic hydroxyl group ofTyr selected from the group consisting of tetrahydropyranyl, tert-butyl,trityl, benzyl, Z, 2-bromobenzyloxycarbonyl(2BrZ) and2,6-dichlorobenzyl(DCB). 2BrZ is preferred.

X⁵ is a protecting group for a side chain guanidino group, such as thatin Arg or Har, or for the imidazole group of His, such as nitro, Tos,trityl, adamantyloxycarbonyl, Z and 2,4-dinitrophenol(Dnp), or X⁵ may behydrogen, which means there is no protection on the side chain groupatoms. Tos is generally preferred.

X⁶ is a protecting group for an amino side chain group, primary orsecondary amino, such as Z or 2ClZ; X^(a) is a subclass of X⁶ comprisingsuch protecting groups that can be removed without removing other sidechain protecting groups so as to allow the omega-amino group tothereafter take part in the reactions to build the unnatural amino-acidresidue. Preferably a base-labile group, such as Fmoc,methylsulfonylethyloxycarbonyl(Msc) or trifluoroacetyl(Tfa), is used;however, it may also be possible to use a hydrazine-labile group such asphthaloyl, ##STR47## or a thiolabile group such as Nps or Dts.

X⁷ is hydrogen or a protecting group for Met, such as oxygen; Met isgenerally left unprotected.

X⁸ may be Gly-NH- resin support!, D-Ala-NH- resin support! or N(A)-resin support!; X⁸ may also be an amide or an ester protecting group orthe like (when solution synthesis is used) either of Gly or of D-Ala ora substituted amide attached directly to Pro or NHNHCONH₂.

The criterion for selecting side chain protecting groups for X² -X⁷ isthat the protecting group should be stable to the reagent under thereaction conditions selected for removing the α-amino protecting group(preferably Boc) at each step of the synthesis. Protecting groupsgenerally should not be split off under coupling conditions but shouldbe removable upon completion of the synthesis of the desired amino acidsequence under reaction conditions that will not alter the peptidechain.

When the X⁸ group is Gly--NH-- resin support! or D--Ala--NH-- resinsupport!, an amide bond connects Gly or D--Ala to a BHA resin or to aMBHA resin. When the X⁸ group is N(A)- resin support!, a substitutedamide bond connects Pro to an N-alkylaminomethyl (NAAM) resin. When X⁸is AzaGly--NH₂, the peptide is preferably made by classical solutionsynthesis, as disclosed in U.S. Pat. No. 4,234,571.

When G is acetyl, for example, in the final formula, a reaction ispreferably carried out with the peptide on the resin (after deblockingthe α-amino group while the side-chain groups remain protected), e.g. byreacting with acetic acid in the presence of diisopropyl or dicyclohexylcarbodiimide (DIC or DCC) or preferably with acetic anhydride. Othersuitable reactions as known in the art can also be used.

Thus, the invention also provides a method for making a peptide, saidpeptide having the formula:

G-AA-AA₂ -AA'-Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀, wherein at least one ofAA', AA₅, AA₆ and AA₈ is U* and the symbols are as set forthhereinbefore, which method comprises (a) forming an intermediate peptidehaving the formula: X¹ -AA-AA₂ (X⁵)-U₃ -Ser(X³)-U₅ -U₆ -AA₇ (X² orX⁷)-U₈ -Pro-X⁸ wherein; U₃ is either U' or AA' (X²); U₅ is either U' orAA₅ (X⁴ or X⁵); U₆ is either U' or AA₆ (X⁴ or X⁵ or X⁶); U₈ is either U'or AA₈ (X⁵ or X⁶); U' is either Lys(X^(a)), Aph(X^(a)), Orn(X^(a)),Dbu(X^(a)) or Dpr(X^(a)); X¹ is hydrogen or an α-amino protecting group;X² is hydrogen or a protecting group for an indole nitrogen; X³ is aprotecting group for a hydroxyl group of Ser or Thr; X⁴ is hydrogen or aprotecting group for a phenolic hydroxyl group of Tyr; X⁵ is eitherhydrogen or a protecting group for a guanidino or imidazole side chain;X⁶ is a protecting group for a primary amino side chain of which X^(a)is a subgroup that is removable without removing other protectinggroups; X⁷ is hydrogen or a protecting group for Met; X⁸ is Gly-NH-resin support!, D-Ala-NH- resin support!, N(A)- resin support!, an amideeither of Gly or of D-Ala or a substituted amide attached directly toPro or NHNHCONH₂ ; provided however that at least one of U₃, U₅, U₆ andU₈ is either Lys(X^(a)), Aph(X^(a)), Orn(X^(a)), Dbu(X^(a)) orDpr(X^(a)); (b) removing at least one X^(a) to deprotect a side chainprimary amino group of at least one amino acid residue of saidintermediate peptide; (c) reacting said deprotected side chain primaryamino group to build said residue into one having the formula U*; and(d) splitting off any remaining groups X¹ to X⁷ and/or cleaving from anyresin support included in X⁸.

Purification of the peptide is effected by ion exchange chromatographyon a CMC column, followed by partition chromatography using the elutionsystem: n-butanol;0.1N acetic acid (1:1 volume ratio) on a column packedwith Sephadex G-25, or by using HPLC, as known in the art andspecifically set forth in J. Rivier, et al. J. Chromatography, 288(1984) 303-328.

The antagonists of the invention are effective at levels of less than100 micrograms per kilogram of body weight, when administeredsubcutaneously at about noon on the day of proestrus, to preventovulation in female rats. For prolonged suppression of ovulation, it maybe necessary to use dosage levels in the range of from about 0.1 toabout 2.5 milligrams per kilogram of body weight. These analogs areparticularly soluble at physiological pHs and thus can be prepared asrelatively concentrated solutions for administration. The antagonistsare also effective to arrest spermatogenesis when administered to malemammals on a regular basis and can thus be used as contraceptives. Sincethese compounds will reduce testosterone levels (an undesiredconsequence in the normal, sexually active male), it may be reasonableto administer replacement dosages of testosterone along with the GnRHantagonist. These antagonists can also be used to regulate theproduction of gonadotropins and sex steroids for other purposes asindicated hereinbefore.

In the following formulas, the U* residues are defined in terms of theoriginal amino acid residue having a side chain amino group plus themodification in question which is set forth in the accompanyingparentheses. Preferably, the original residue is incorporated in themain peptide chain, for example Lys or D-Lys or Orn, Dbu, Dpr or aD-isomer thereof, and is modified while a part of the peptide chain thatis still attached to the resin to form the desired residue of the aminoacid U*. However, as indicated hereinbefore, the suitably protectedunnatural amino acid U* can be added as a part of the usual chainelongation process.

With respect to modified side chain amino groups of the amino acids Lys,Orn, Dbu and Dpr, the following abbreviations are used:

act=3-acetylamino 1,2,4 triazole

atz=3-amino 1,2,4 triazole (alternatively referred to as tcg)

bcg=aminobutyl cyanoguanidino

bzcg=aminobenzyl cyanoguanidino

bur=N-g-amido, N-g'-butylguanidino

chcg=aminocyclohexyl cyanoguanidino

ecg=aminoethyl cyanoguanidino

icg=aminoisopropyl cyanoguanidino

hcg=aminohexyl cyanoguanidino

hicg=histaminyl cyanoguanidino (ethylimidazole)

mcg=aminomethyl cyanoguanidino

ncg=aminoethyl(1 or 2)naphthyl cyanoguanidino

mncg=aminomethyl(1 or 2)naphthyl cyanoguanidino

Ocg=O-phenyl cyanoguanidino

pcg=aminopropyl cyanoguanidino

Sbcg=thiobutyl cyanoguanidino

trcg=indole ethylamino cyanoguanidino(tryptamino cyanoguanidino)

mpcg=aminomethyl pyridyl cyanoguanidino (number indicates position ofaminomethyl group on pyridyl ring)

The following Examples illustrate a large number of peptides embodyingvarious features of the invention. All of these peptides include atleast one D-isomer amino acid residue.

EXAMPLE I

Peptides as indicated in TABLE I having the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ areprepared by the solid-phase procedure referred to above.

                  TABLE I                                                         ______________________________________                                                AA.sub.5  AA.sub.6    AA.sub.8                                        ______________________________________                                        1         Lys(icg)    D--Lys(icg) ILys                                        2         Lys(mcg)    D--Lys(mcg) ILys                                        3         Lys(chcg)   D--Lys(chcg)                                                                              ILys                                        4         Lys(atz)    D--Lys(atz) ILys                                        5         Lys(pcg)    D--Lys(pcg) ILys                                        6         Lys(2mpcg)  D--Lys(2mpcg)                                                                             ILys                                        7         Lys(3mpcg)  D--Lys(3mpcg)                                                                             ILys                                        8         Lys(4mpcg)  D--Lys(4mpcg)                                                                             ILys                                        9         Lys(hcg)    D--Lys(hcg) ILys                                        10        Lys(ecg)    D--Lys(ecg) ILys                                        11        Lys(Ocg)    β-D--2NAL                                                                            ILys                                        12        Lys(bcg     β-D--2NAL                                                                            ILys                                        13        Lys(Nic)    D--Lys(Nic) Lys(bcg)                                    14        Lys(bcg)    β-D--2NAL                                                                            Lys(bcg)                                    15        Lys(bcg)    D--Lys(bcg) Arg                                         16        Tyr         β-D--2NAL                                                                            Lys(icg)                                    ______________________________________                                    

For purposes of an example, a representative solid phase synthesis ofPeptide No. 1 above, which is referred to as Ac-β-D-2NAL¹, (4Cl)D-Phe²,D-3PAL³, LYs(icg)⁵, D-Lys(icg)⁶, ILys⁸, D-Ala¹⁰ !-GnRH is set forthhereinafter. This peptide has the following formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Lys(isopropylcyanoguanidino)-D-Lys(isopropylcyanoguanidino)-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂.

An MBHA resin is used, and Boc-protected D-Ala is coupled to the resinover a 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivativeand DCC as an activating reagent. The D-Ala residue attaches to the MBHAresidue by an amide bond.

Following the coupling of each amino acid residue, washing, deblockingand coupling of the next amino acid residue is carried out in accordancewith the following schedule using an automated machine and beginningwith about 5 grams of resin:

    ______________________________________                                                                        MIX                                                                           TIMES                                         STEP REAGENTS AND OPERATIONS    MIN.                                          ______________________________________                                        1    CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                                  3                                             2    Methanol (MeOH) wash-30 ml. (2 times)                                                                    3                                             3    CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                             4    50 percent TFA plus 5 percent 1,2-ethanedithiol                                                          10                                                 in CH.sub.2 Cl.sub.2 -70 ml. (2 times)                                   5    Isopropyl alcohol + 1% ethanedithiol wash-80 ml.                                                         3                                                  (2 times)                                                                6    TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml. (2 times)                                                  5                                             7    MeOH wash-40 ml. (2 times) 2                                             8    CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                             9    Boc-amino acid (10 mmoles) in 30 ml. of either di-                                                        30-300                                            methylformamide (DMF) or CH.sub.2 Cl.sub.2, depending upon                    the solubility of the particular protected amino acid,                        (1 time) plus DIC or DCC (10 mmoles) in CH.sub.2 Cl.sub.2                10   MeOH wash-40 ml. (2 times) 3                                             11   Triethylamine (TEA) 12.5 percent in CH.sub.2 Cl.sub.2 -70                                                3l.                                                (1 time)                                                                 ______________________________________                                    

After step 3, an aliquot may be taken for a ninhydrin test as well knownin the art: if the test is negative, proceed to step 4 for removal ofBOC-group prior to coupling of the next amino acid; if the test ispositive or slightly positive, repeat steps 9 through 11.

The above schedule is used for coupling of each of the amino acids ofthe peptide of the invention after the first amino acid has beenattached. N.sup.α Boc protection is used for each of the remaining aminoacids throughout the synthesis. N.sup.α Boc-β-D-2NAL is prepared by amethod known in the art, e.g. as described in detail in U.S. Pat. No.4,234,571, issued Nov. 18, 1980 or commercially available fromSyntheTech, Oregon, U.S.A. The side chains of Lys in the 5-position andof D-Lys in the 6-position are protected with Fmoc. Bzl (benzyl ether)is used as a side chain protecting group for the hydroxyl group of Ser.Boc-Lys(Ipr) is used for the 8-position. After deblocking the α-aminogroup at the N-terminal using trifluoroacetic acid(TFA), acetylation isachieved using a large excess of acetic anhydride in dichloromethane.

Following completion of the assembly of the peptide and acetylation ofthe N-terminus, the following intermediate is present:Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Lys(Fmoc)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-MBHA resin support!. The unnatural amino acids in the 5- and 6-positionsare formed by simultaneously carrying out the following reactions withthe deprotected side chains of the Lys residues. The Fmoc protectinggroup is removed from both by treatment of the peptidoresin with 20percent piperidine in DMF for 5 minutes, then washing with DMF, thentreatment with more piperidine/DMF for 20 minutes. After washing theresin with DMF, CH₃ OH, CH₂ Cl₂, and finally DMF, the newly freed aminogroup is treated with a large excess (>10 fold) of diphenylcyanocarbonimidate(PCI) in DMF. Thereafter, the peptide is thensubjected to the standard wash (see Steps 10-11) and then treated withisopropylamine dissolved in DMF for 24 hours at about 22° C. to completethe formation of the aminoisopropyl cyanoguanidino moiety; for some ofthe more hindered reactants, this step may be repeated.

The cleavage of the peptide from the resin and deprotection of the Serside chain takes place very readily at 0° C. with HF. Anisole is addedas a scavenger prior to HF treatment. After the removal of HF undervacuum, the resin is extracted with 50% acetic acid, and the washingsare lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by high performance liquidchromatography (HPLC), as known in the art and specifically set forth inJ. Rivier, et al. J. Chromatography, 288 (1984) 303-328.

The peptide is judged to be homogeneous using thin layer chromatographyand several different solvent systems, as well as by usingreversed-phase high pressure liquid chromatography and an aqueoustriethylammonium phosphate solution plus acetonitrile. Amino acidanalysis of the resultant, purified peptide is consistent with theformula for the prepared structure, showing substantially integer-valuesfor each amino acid in the chain; mass spectral analysis is alsoconsistent. The optical rotation is measured on a photoelectricpolarimeter as

     α!.sup.20 .sub.D =-2.8±0.5(c=1, 50% acetic acid).

The other peptides in Table I are similarly synthesized and purified.The peptides are assayed in vivo and may also be tested in vitro. Ifperformed, in vitro testing is carried out using dissociated ratpituitary cells maintained in culture for 4 days prior to the assay. Thelevels of LH mediated in response to the application of peptides isassayed by specific radioimmunoassay for rat LH. Control dishes of cellsonly receive a measure which is 3 nanomolar in GnRH; experimental dishesreceive a measure 3 nanomolar in GnRH plus a measure having either thepresent standard antagonist for comparison purposes i.e. Ac-dehydroPro¹, (4F)D-Phe², D-Trp³,6 !-GnRH or the test peptide, in concentrationsranging from 0.01 to 10 nanomolar. The amount of LH secreted in thesamples treated only with GnRH is compared with that secreted by thesamples treated with the peptide plus GnRH. The ability of the testpeptide to reduce the amount of LH released by 3 nanomolar GnRH iscompared to that of the present standard peptide.

The in vivo testing determines effectiveness to prevent ovulation infemale rats. In this test, a specified number of mature femaleSprague-Dawley rats, e.g. five to ten, each having a body weight from225 to 250 grams, is injected with a specified microgram dosage ofpeptide in either saline, bacteriostatic water, polyethylene glycol,corn oil or mixtures of the above with ethanol at about noon on the dayof proestrus. Proestrus is the afternoon of ovulation. A separate femalerat group is used as a control to which the peptide is not administered.Each of the control female rats ovulates on the evening of proestrus; ofthe rats treated, the number of them which ovulate is recorded. Each ofthe peptides is considered to be totally effective to prevent ovulationof female rats at a dose of about 500 micrograms.

All peptides listed in Table I are considered effective to blockGnRH-induced LH secretion in vitro at some reasonable concentration. Allof the peptides are considered to be effective to prevent ovulation offemale mammals at low dosages. The following Table A shows the resultsof in vivo testing of various of these GnRH antagonists with the dosagesbeing given in micrograms:

                  TABLE A                                                         ______________________________________                                        Peptide          Rats              Rats                                       No.      Dosage  Ovulating   Dosage                                                                              Ovulating                                  ______________________________________                                        1.       2.5     0/9         1.0   0/5                                        2.       2.5     6/6         2.0   2/10                                       3.       2.5     7/9         2.0   1/10                                       4.       2.5     0/6         2.0   1/10                                       5.       2.0      1/10                                                        6.       2.5     0/6         1.0   7/17                                       7.       2.5      3/20                                                        8.       2.5      5/19       1.0   5/5                                        9.       2.5     0/6         1.0   5/5                                        10.      2.5     5/8         2.0   1/10                                       12.      2.5     6/7         5.0   9/10                                       13.      5.0      5/12       10.0  3/5                                        15.      2.5     0/4         1.0   8/18                                       ______________________________________                                    

EXAMPLE II

Peptides as indicated in TABLE II having the formula:Ac-dehydroPro-(A)D-Phe-AA₃ -Ser-AA₅ -β-D-2NAL-Leu-AA₈ -Pro-D-Ala-NH₂ areprepared by the solid-phase procedure referred to above.

                  TABLE II                                                        ______________________________________                                        A          AA.sub.3    AA.sub.5   AA.sub.8                                    ______________________________________                                        17   4Cl       β-D--2NAL                                                                            Tyr      Lys(bcg)                                  18    "          "          "       Lys(ecg)                                  19   4F        (1For)D--Trp                                                                              (2F)Phe  Orn(ecg)                                  20    "          "         Tyr      Dpr(2ncg)                                 21    "          "         (2NO.sub.2)Phe                                                                         Dbu(icg)                                  22    "        (1Ac)D--Trp (2CH.sub.3)Phe                                                                         Dbu(2mpcg)                                23   4Br         "         Tyr      Dbu(3mpcg)                                24    "          "         (2Br)Phe Dbu(4mpcg)                                25   H         D--Trp      (2Cl)Phe Orn(2mncg                                 26   4NO.sub.2 (5CH.sub.3)D--Trp                                                                         (3CH.sub.3)Phe                                                                         Orn(hcg)                                  27    "        (5F)D--Trp  His      Lys(atz)                                  28   2,4Cl.sub.2                                                                             (5Cl)D--Trp (3F)Phe  Dpr(trcg)                                 29    "        (6NO.sub.2)D--Trp                                                                         (3Br)Phe Orn(1ncg)                                 30   C.sup.α Me/4Cl                                                                    (50CH.sub.3)D--Trp                                                                        (3I)Tyr  Orn(pcg)                                  31   3,4Cl.sub.2                                                                             (5NH.sub.2)D--Trp                                                                         (Cl)Phe  Dpr(chcg)                                 ______________________________________                                    

All peptides listed in Table II are considered effective to blockGnRH-induced LH secretion in vitro at some reasonable concentration. Allof the peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

EXAMPLE III

Peptides as indicated in TABLE III having the formula:G-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Tyr-AA₆ -Leu-AA₈ -Pro-AA₁₀ are preparedby the solid-phase procedure referred to above.

                  TABLE III                                                       ______________________________________                                        G         AA.sub.6    AA.sub.8   AA.sub.10                                    ______________________________________                                        32    Ac      D--Arg      Lys(bcg) D--Ala--NH.sub.2                           32A   Ac      D--Arg      Lys(atz) D--Ala--NH.sub.2                           33    Ac      D--Lys(bcg) Arg        "                                        34    For     D--Tyr      Lys(icg) Gly--NH.sub.2                              35    Bz      (Et)D--Arg  Orn(icg)   "                                        36    Ac      D--Lys      Orn(ecg)   "                                        37    Vac     D--Har      Orn(mcg)   "                                        38    Acr     (4gua)D--Phe                                                                              Dpr(2ncg)                                                                              AzaGly--NH.sub.2                           39    Ac      D--Orn      Dpr(chcg)                                                                              D--Ala--NH.sub.2                           40    Acr     D--His      Dpr(atz)   "                                        41    Ac      (Bu)D--Arg  Dbu(1mncg)                                                                               "                                        42     "      (Bz)D--Orn  Dbu(2mpcg)                                                                               "                                        43    Vac     (4NH.sub.2)D--Phe                                                                         Dbu(4mpcg)                                                                               "                                        44    Bz      (Ac)D--Lys  Dbu(trcg)                                                                              AzaGly--NH.sub.2                           ______________________________________                                    

All peptides listed in Table III are considered effective to blockGnRH-induced LH secretion in vitro at some reasonable concentration. Allof the peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

EXAMPLE IV

Peptides as indicated in TABLE IV having the formula: Ac-AA₁-(4Cl)D-Phe-D-3PAL-Ser-Tyr-D-Arg-AA₇ -AA₈ -Pro-D-Ala-NH₂ are prepared bythe solid-phase procedure referred to above.

                  TABLE IV                                                        ______________________________________                                        AA.sub.1          AA.sub.7    AA.sub.8                                        ______________________________________                                        45      β-D--2NAL                                                                              Leu         Lys(bcg)                                    46      (1AC)D--Trp   Met         Lys(2mpcg)                                  47      (6Br)D--Trp   Tyr         Lys(3mpcg)                                  48      (5F)D--Trp    Nle         Lys(4mpcg)                                  49      (6NO.sub.2)D--Trp                                                                           Met         Orn(chcg)                                   50      (5Cl)D--Trp   Tyr         Orn(pcg)                                    51      (4Cl)D--Phe   Phe         Dpr(pcg)                                    52      (4F)D--Phe    (4F)Phe     Dpr(atz)                                    53      (2,4Cl.sub.2)D--Phe                                                                         NML         Dbu(mcg)                                    54      dehydroPro    Nle         Dbu(2ncg)                                   55      β-D--2NAL                                                                              Leu         Lys(Ocg)                                    56      (60CH.sub.3)D--Trp                                                                          (1For)Trp   Orn(1ncg)                                   57      (5NH.sub.2)D--Trp                                                                           Nva         Orn(ecg)                                    58      (4NO.sub.2)D--Phe                                                                           NML         Orn(1mncg)                                  59      dehydroPro    (6NO.sub.2)Trp                                                                            Dbu(hicg)                                   ______________________________________                                    

All peptides listed in Table IV are considered effective to blockGnRH-induced LH secretion in vitro at some reasonable concentration. Allof the peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

EXAMPLE V

Peptides as indicated in TABLE V having the formula: Ac-AA₁-(4Cl)D-Phe-AA₃ -Ser-AA₅ -AA₆ -Leu-ILys-Pro-D-Ala-NH₂ are prepared bythe solid-phase procedure referred to above.

                  TABLE V                                                         ______________________________________                                        AA.sub.1   AA.sub.3   AA.sub.5  AA.sub.6                                      ______________________________________                                        60  β-D--2NAL                                                                           D--3PAL    Lys(bcg)                                                                              β-D--2NAL                              61    "        (6NO.sub.2)D--Trp                                                                          "     (Dnp)D--His                                 62    "        D--Trp     Lys(ecg)                                                                              (4gua)D--Phe                                63  dehydroPro β-D--2NAL                                                                           Orn(ecg)                                                                              (6NO.sub.2)D--Trp                           D--1NAL        β                                                             Orn(mcg)   D--Val                                                         65  β-D--2NAL                                                                           (1For)D--Trp                                                                             Orn(atz)                                                                              (Pr)D--Arg                                  66    "          "        Dbu(bcg)                                                                              (5NH.sub.2)D--Trp                           67  dehydroPro D--Trp     Dbu(2mpcg)                                                                            D--Tyr                                      68    "        D--2PAL    Dbu(4mpcg)                                                                            D--Nle                                      69    "        (1Ac)D--Trp                                                                              Dbu(hcg)                                                                              (4F)D--Phe                                  70  Pro        D--3PAL    Lys(pcg)                                                                              β-D--1NAL                              71  (1For)D--Trp                                                                               "        Lys(chcg)                                                                             (4NHCH.sub.3)D--Phe                         72  β-D--2NAL                                                                             "        Dpr(hcg)                                                                              (Ac)D--Orn                                  73    "          "        Dpr(Ocg)                                                                              (4NH.sub.2)D--Phe                           74  β-D--1NAL                                                                           (6Br)D--Trp                                                                              Dpr(atz)                                                                              (1For)D--Trp                                75  (6CH.sub.3)D--Trp                                                                        D--4PAL    Dpr(bzcg)                                                                             D--4PAL                                     ______________________________________                                    

The peptides listed in Table V are considered effective to blockGnRH-induced LH secretion in vitro at a reasonable concentration. All ofthe peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

EXAMPLE VI

Peptides as indicated in TABLE VI having the formula: G-AA₁-(4Cl)D-Phe-D-Trp-Ser-Tyr-AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ are prepared bythe solid-phase procedure referred to above.

                  TABLE VI                                                        ______________________________________                                        G          AA.sub.1   AA.sub.6    AA.sub.8                                    ______________________________________                                        76     Ac      dehydroPro D--Lys(bcg)                                                                             Lys(bcg)                                  77     Ac      β-D--2NAL                                                                           β-D--2NAL                                                                          Lys(3mpcg)                                78     Ac      β-D--2NAL                                                                           D--Val    Lys(atz)                                  79     Acr     Pro        D--Ser(OtBu)                                                                            Lys(chcg)                                 80     H       dehydroPro (imBzl)D--His                                                                           Lys(ecg)                                  81     Bz      (4Br)D--Phe                                                                              (5Cl)D--Trp                                                                             Orn(ecg)                                  82      "      D--pGlu    (6Br)D--Trp                                                                             Orn(bzcg)                                 83     For     β-D--1NAL                                                                           (Me)D--Arg                                                                              Orn(Ocg)                                  84      "      dehydroPro D--Har    Orn(4mpcg)                                85     Vac     β-D--2NAL                                                                           (Bz)D--Lys                                                                              Lys(chcg)                                 86     Ac      β-D--(Arg.sup.5)                                                                    β-D--2NAL                                                                          Lys(Ocg)                                  87     H       dehydroPro D--Ala    Lys(atz)                                  ______________________________________                                    

The peptides listed in Table VI are considered effective to blockGnRH-induced LH secretion in vitro at a reasonable concentration. All ofthe peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

EXAMPLE VII

Peptides as indicated in TABLE VII having the formula:Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ areprepared by the solid-phase procedure referred to above.

                  TABLE VII                                                       ______________________________________                                                AA.sub.5 AA.sub.6     AA.sub.8                                        ______________________________________                                        88        Arg        D--Lys(bcg)  Arg                                         89         "         D--Lys(Ocg)   "                                          90        Orn(bcg)   (4gua)D--Phe (Et.sub.2)Arg                               91        Lys(2ncg)  D--Lys(2ncg) ILys                                        92        Orn(bzcg)  D--Lys(bzcg) Har                                         93        Lys(act)   D--Lys(act)  ILys                                        94        Lys(hicg)  D--Lys(hicg) ILys                                        95        Lys(trcg)  D--Lys(trcg) ILys                                        96        Lys(bcg)   D--Lys(bcg)  ILys                                        97        Lys(bcg)   D--3PAL      (EtPr)Har                                   98        Lys(bcg)   D--Lys(Nic)  (Me.sub.2)Arg                               99        Orn(atz)   D--Lys(atz)  (MeBu)Arg                                   100       Lys(Sbcg)  D--Lys(Sbcg) ILys                                        ______________________________________                                    

The peptides listed in Table VII are considered effective to blockGnRH-induced LH secretion in vitro at a reasonable concentration. All ofthe peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

EXAMPLE VIII

Peptides as indicated in TABLE VIII having the formula:Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ areprepared by the solid-phase procedure referred to above.

                  TABLE VIII                                                      ______________________________________                                               AA.sub.5  AA.sub.6      AA.sub.8                                       ______________________________________                                        101      Orn(bcg)    D--Orn(bcg)   ILys                                       101A     Lys(bcg)    D--Lys(bcg)   ILys                                       102      Orn(2mpcg)  D--Orn(2mpcg) ILys                                       103      Orn(atz)    D--Orn(atz)   ILys                                       103A     Aph(atz)    D--Aph(atz)   ILys                                       103B     Lys(atz)    D--Lys(atz)   ILys                                       104      Orn(bcg)    β-D--2NAL                                                                              ILys                                       104A     Aph(bcg)    D--Aph(bcg)   ILys                                       105      Orn(2mpcg)  β-D--2NAL                                                                              ILys                                       105A     Lys(Nic)    D--Lys(Nic)   Lys(atz)                                   106      Lys(Nic)    D--Lys(tcg)   Lys(bur)                                   106A     Lys(atz)    D--Lys(Nic)   ILys                                       106B     Lys(Nic)    D--Lys(tcg)   ILys                                       107      Lys(2ncg)   D--Lys(2ncg)  ILys                                       108      Lys(bzcg)   D--Lys(bzcg)  ILys                                       109      Lys(icg)    D--Lys(icg)   Arg                                        110      Orn(icg)    D--Orn(icg)   ILys                                       ______________________________________                                    

As a further working example, a solid phase synthesis is set forthhereinafter of Peptide No. 103A above, which is referred to asAc-β-D-2NAL¹, (4Cl)D-Phe², D-3PAL³, Aph(atz)⁵, D-Aph(atz)⁶, ILys⁸,D-Ala¹⁰ !-GnRH. This peptide has the following formula:

Ac-β-D-2NAL-(4Cl) D-Phe-D-3PAL-Ser-Aph(3-amino 1,2,4triazole)-D-Aph(3-amino 1,2,4triazole)-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂.

An MBHA resin is used, and Boc-protected D-Ala is coupled to the resinover a 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivativeand DCC as an activating reagent. The D-Ala residue attaches to the MBHAresidue by an amide bond.

Following the coupling of each amino acid residue, washing, deblockingand coupling of the next amino acid residue is carried out in accordancewith the schedule set forth hereinbefore, using an automated machine andbeginning with about 5 grams of resin. The side chain primary aminogroups of Aph in the 5-position and of D-Aph in the 6-position areprotected by Fmoc. Bzl(benzyl ether) is used as a side chain protectinggroup for the hydroxyl group of Ser. Boc-Lys(Ipr,Z) is used for the8-position. After deblocking the α-amino group at the N-terminus usingtrifluoroacetic acid (TFA), acetylation is achieved using a large excessof acetic anhydride in dichloromethane.

Following completion of the assembly of the peptide and acetylation ofthe N-terminus, the following intermediate is present:Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Aph(Fmoc)-D-Aph(Fmoc)-Leu-Lys(Ipr,Z)-Pro-D-Ala-NH-MBHA resin support!. The unnatural amino acids in the 5- and 6-positionsare formed by simultaneously carrying out the following reactions withthe deprotected side chains of the Aph residues. The Fmoc protectinggroup is removed from both by treatment of the peptidoresin with 20percent piperidine in DMF for 5 minutes, then washing with DMF, thentreatment with more piperidine/DMF for 20 minutes. After washing theresin with DMF, CH₃ OH, CH₂ Cl₂, and finally DMF, the newly freed aminogroup is treated with a large excess (>10 fold) of diphenylcyanocarbonimidate(PCI) in DMF. Thereafter, the peptide is thensubjected to the standard wash and then treated with hydrazine,dissolved in DMF, for 24 hours at about 22° C. to complete the formationof the cyanoguanidino moiety; this step is preferably repeated. Thecyanoguanidino moieties that are formed spontaneously convert to thecorresponding heterocycle, i.e. 3-amino, 1,2,4 triazole.

The cleavage of the peptide from the resin and deprotection of the Serand the Lys side chains takes place very readily at 0° C. with HF.Anisole is added as a scavenger prior to HF treatment. After the removalof HF under vacuum, the resin is extracted with 50% acetic acid, and thewashings are lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by high performance liquidchromatography (HPLC), as known in the art and specifically set forth inJ. Rivier, et al. J. Chromatography, 288 (1984) 303-328.

The peptide is judged to be homogeneous using capillary zoneelectrophoresis (CZE), as well as by using reversed-phase high pressureliquid chromatography and an aqueous triethylammonium phosphate solutionplus acetonitrile. Amino acid analysis of the resultant, purifiedpeptide is consistent with the formula for the prepared structure,showing substantially integer-values for each amino acid in the chain;mass spectral analysis is also consistent. The optical rotation ismeasured on a photoelectric polarimeter as

     α!.sup.20.sub.D =-33±1.0(c=1, 50% acetic acid).

In addition to the above synthesis, Peptide 103A is also synthesizedwith glycinamide at the C-terminus instead of D-Ala-NH₂ ; biologicaltesting in vivo shows it is slightly less biologically potent thanPeptide 103A.

Peptide 103A was also synthesized with the N-ethylamide at theC-terminus instead of D-Ala-NH₂ ; biological testing in vivo showed itexhibited just slightly less biological potency than that reported inTABLE B for Peptide 103B. In addition, Peptide 104A is also synthesizedwith the glycinamide at the C-terminus instead of D-Ala-NH₂ ; biologicaltesting in vivo shows somewhat less biological potency than thatreported in TABLE B for Peptide 104A.

All peptides listed in Table VIII are considered effective to blockGnRH-induced LH secretion in vitro at some reasonable concentration. Allof the peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

In addition to the in vivo testing, mentioned above, to determineeffectiveness to prevent ovulation in female rats, groups of about 6adult castrate male Sprague-Dawley rats, each having a body weight from225 to 250 grams, are each injected IV with a 50 microgram dosage ofeither Peptide No. 103A or a standard antagonist referred to as theNal-Glu antagonist (see J. Clin. Endo. Metab., 71, 4, 881-888 (1990)) ineither corn oil or a phosphate buffer in the presence of BSA (bovineserum albumin). A separate rat group is used as a control, to which onlythe carrier is administered. Each of the control rats and the rats beingtreated are monitored for LH levels in the bloodstream at about 3 hours,24 hours, 36 hours and 48 hours following this single bolus injection.Peptide No. 103A is surprisingly effective to suppress the level ofcirculating LH in the bloodstream of male rats, at levels verysubstantially below that of the control and, after 12 hours, below thatof the rats treated with the Nal-Glu antagonist. This very long-actingduration of biological effect was truly surprising.

EXAMPLE IX

Peptides as indicated in TABLE IX having the formula:Ac-β-D-2NAL-(4Cl)D-Phe-AA₃ -Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ areprepared by the solid-phase procedure referred to above.

                  TABLE IX                                                        ______________________________________                                        AA.sub.3       AA.sub.5  AA.sub.6   AA.sub.8                                  ______________________________________                                        111   D--Lys(bcg)  Tyr       D--Arg   Arg                                     111A  D--Aph(icg)  Tyr       D--3PAL  ILys                                    112   D--Lys(2mpcg)                                                                              Tyr       D--Arg   Arg                                     113   D--Lys(atz)  Tyr       D--Arg   Arg                                     113A  D--Lys(atz)  Tyr       D--Lys(atz)                                                                            ILys                                    114   D--Lys(bcg)  Arg       β-D--2NAL                                                                         Arg                                     114A  D--Lys(2mpcg)                                                                              Arg       β-D--2NAL                                                                         Arg                                     115   D--Lys(atz)  Arg       β-D--2NAL                                                                         Arg                                     116   D--Lys(bcg)  Tyr       D--Arg   ILys                                    117   D--Lys(atz)  Tyr       D--Arg   ILys                                    117A  D--Lys(atz)  Tyr       D--3PAL  Arg                                     117B  D--Lys(atz)  Tyr       D--3PAL  ILys                                    118   D--Lys(bcg)  Tyr       β-D--2NAL                                                                         ILys                                    119   D--Lys(bcg)  Tyr       D--3PAL  ILys                                    119A  D--Aph(bcg)  Tyr       D--3PAL  ILys                                    120   D--Lys(2mpcg)                                                                              Tyr       D--3PAL  ILys                                    120A  D--Lys(2mpcg)                                                                              Arg       β-D--2NAL                                                                         Arg                                     121   D--Lys(bcg)  Tyr       D--Arg   Arg                                     122   D--Lys(bcg)  Lys(bcg)  D--Lys(bcg)                                                                            ILys                                    122A  D--Lys(bcg)  Tyr       D--Lys(bcg)                                                                            ILys                                    123   D--3PAL      Lys(bur)  β-D--2NAL                                                                         Arg                                     124   D--Lys(bcg)  Tyr       β-D--2NAL                                                                         Lys(bcg)                                125   D--Orn(2mpcg)                                                                              Tyr       β-D--2NAL                                                                         ILys                                    125A  D--Orn(atz)  Tyr       D--3PAL  ILys                                    135B  D--Orn(bcg)  Tyr       D--3PAL  ILys                                    ______________________________________                                    

All peptides listed in Table IX are considered effective to blockGnRH-induced LH secretion in vitro at some reasonable concentration. Allof the peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

EXAMPLE X

Peptides as indicated in TABLE X having the formula:Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -AA₇ -ILys-Pro-AA₁₀ areprepared by the solid-phase procedure referred to above.

                  TABLE X                                                         ______________________________________                                        AA.sub.5     AA.sub.6     AA.sub.7                                                                              AA.sub.10                                   ______________________________________                                        125C  Aph(atz)   D--Aph(atz)  NML   D--Ala--NH.sub.2                          125D    "          "          Leu   NHCH.sub.2 CH.sub.3                       125E    "          "          NML     "                                       125F    "          "          Leu   Gly--NH.sub.2                             125G  Aph(2mpcg) D--Aph(2mpcg)                                                                              NML   D--Ala--NH.sub.2                          ______________________________________                                    

The peptides listed in Table X are considered effective to blockGnRH-induced LH secretion in vitro at a reasonable concentration. All ofthe peptides are considered to be effective to prevent ovulation offemale mammals at low dosages.

Results of in vivo testing of selected of these antagonists are shown inthe following Table B, with the dosages being given in micrograms:

                  TABLE B                                                         ______________________________________                                        Peptide          Rats              Rats                                       No.      Dosage  Ovulating   Dosage                                                                              Ovulating                                  ______________________________________                                        17.      10      6/8         15     7/10                                      32A.     10      0/6         5     0/6                                                 2.5     0/7         1.0    5/10                                      33.      2.5      4/17                                                        55.      10      6/7         25    2/5                                        60.      2.5      4/14       1.0    8.10                                      86.      2.5      4/15       5.0   5/5                                        88.      2.5     0/4         1.0    5/15                                               10      0/8                                                          89.      10       2/18                                                        93.      2.5      5/10                                                        94.      1.0      7/11                                                        96.      1.0      9/12                                                        101.     2.5     0/5         1.0   5/8                                        102.     2.5     2/9                                                          103.     2.5     5/7                                                          103A.    2.5     0/8         1.0   0/7                                                                     0.5   7/8                                        103B.    2.5     0/7         1.0   3/9                                                                     0.5   7/8                                        104.     2.5     6/8                                                          104A.    5.0      6/11       2.5   2/3                                        105.     2.5     0/5                                                          105A.    5.0     4/6                                                          106.     5.0     0/7                                                          106A.    2.0     0/5         1.0   7/7                                        106B.    2.5     0/6         1.0   4/6                                        111.     2.5      1/11       1.0   0/5                                                                     0.5   5/7                                        111A.    15      0/9         5     2/4                                        112.     1.0     4/8                                                          113.     1.0     5/6                                                          113A     5.0     0/6         2.5    1/15                                      114.     5.0     4/5                                                          115.     2.5     2/8                                                          116.     2.5     0/6         1.0   2/8                                        117.     10      0/6         5     2/5                                        117A.    2.5     0/7         1.0   4/9                                        117B.    15      1/5                                                          118.     15      4/5         5     5/5                                        119.     2.5     0/7         1.0    2/10                                      119A.    15      4/6                                                          120.     2.5     0/8         1.0   6/7                                        120A.    5.0     4/6                                                          122.     10      4/6                                                          122A.    5.0     0/6         2.5   5/6                                        123.     10       1/10       5     0/6                                        125.     2.5     3/7                                                          125A.    5.0     1/8         2.5   1/7                                        125B.    2.5     0/7                                                          125C.    1.0     0/8         0.5    6/10                                      125D.    2.5     0/8         1.0   8/9                                        125E.    10      0/5                                                          125F.    1.0      9/16                                                        125G.    5.0     0/6                                                          ______________________________________                                    

EXAMPLE XI

Peptides as indicated in TABLE XI having the formula:pGlu-His-Trp-Ser-Arg-AA₆ -AA₇ -Arg-Pro-AA₁₀ are prepared by the solidphase procedure referred to above.

                  TABLE XI                                                        ______________________________________                                               AA.sub.6    AA.sub.7 AA.sub.10                                         ______________________________________                                        126      D--Lys(bcg)   Leu      Gly--NH.sub.2                                 127      D--Lys(ecg)    "         "                                           128      D--Lys(pcg)    "         "                                           129      D--Lys(hcg)    "       AzaGly--NH.sub.2                              130      D--Lys(atz)    "       NHCH.sub.2 CH.sub.3                           131      D--Lys(4mpcg)  "         "                                           132      D--Lys(3mpcg) NML        "                                           133      D--Lys(2mpcg)  "         "                                           134      D--Lys(chcg)  Leu      Gly--NH.sub.2                                 135      D--Lys(Ocg)    "         "                                           136      D--Orn(bcg)    "         "                                           137      D--Orn(atz)   NML      AzaGlyNH.sub.2                                138      D--Orn(mcg)    "       NHCH.sub.2 CH.sub.3                           139      D--Dbu(2mpcg)  "       NHCH.sub.3                                    140      D--Dbu(chcg)  Leu      NHCH.sub.2 CH.sub.2 CH.sub.3                  141      D--Dbu(bzcg)   "       Gly--NH.sub.2                                 142      D--Dpr(ecg)    "         "                                           143      D--Dpr(hicg)  NML        "                                           144      D--Dpr(trcg)   "       NHCH.sub.2 CH.sub.3                           ______________________________________                                    

The peptides described in TABLE XI are considered to be effective tocause the release of LH and FSH in female rats. All of them areconsidered to be substantially more effective than native GnRH.

EXAMPLE XII

A peptide intermediate having the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Lys(Fmoc)-D-3PAL-NML-Lys(Dts)-Pro-D-Ala-NH-resin support! is prepared by the solid phase procedure generallyreferred to above. The intermediate is then treated with piperidine toremove the Fmoc protecting group and is thereafter reacted with PCI ashereinbefore described. Next the Dts protecting group is removed fromthe amino side chain of the Lys residue in the 8-position using asuitable thiol, such as β-mercaptoethanol or thiophenol(PhSH), in DMF,and the peptidoresin is given the standard wash. Thereafter, it ismaintained at 22° C. for 10-60 minutes or until the ninhydrin test isnegative to allow the reaction to proceed to completion, effectingcyclization of the side-chain primary amino group of Lys⁸ and thecyanoguanidino moiety which was earlier formed upon the Lys⁵ side chain.Deprotection and cleavage are then carried out as previously described.Following HPLC purification as previously described, the GnRH antagonist(Peptide No. 145) is tested. The peptide is considered to be effectiveto prevent ovulation of female mammals at low dosages.

EXAMPLE XIII

A peptide intermediate having the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Orn(Fmoc)-D-Trp-Leu-Lys(Nps)-Pro-D-Ala-NH-resin support! is prepared by the solid phase procedure referred toabove. The peptide intermediate is treated with a suitable thiol asdescribed in Example XII to form the cyanoguanidino moiety by reactionof PCI with the side chain amino group of the Lys residue in the8-position, and it is then cyclized with the deprotected side chainamino group of Orn in the 5-position following removal of the Fmocprotecting group. Following cleavage and HPLC purification as previouslydescribed, the GnRH antagonist (Peptide No. 146) is tested. The peptideis considered to be effective to prevent ovulation of female mammals atlow dosages.

EXAMPLE XIV

A peptide intermediate having the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-resin support! is prepared by the solid phase procedure referred toabove. Following the removal of the Fmoc protection, the peptideintermediate is reacted as generally described in Example I usingnaphthyl isocyanate instead of PCI to form the napthylurea moiety withthe side chain amino group of the D-Lys residue in the 6-position.Following cleavage and HPLC purification as previously described, theGnRH antagonist (Peptide No. 147) is tested. The peptide is consideredto be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XV

A peptide intermediate having the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-resin support! is prepared by the solid phase procedure referred toabove. Following the removal of the Fmoc protection, the peptideintermediate is reacted as generally described in Example I usingnaphthyl isothiocyanate instead of PCI to form the napthylthioureamoiety with the side chain amino group of the residue in the 6-position.Following cleavage and HPLC purification as previously described, theGnRH antagonist (Peptide No. 148) is tested. The peptide is consideredto be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XVI

A peptide intermediate having the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-resin support! is prepared by the solid phase procedure referred toabove. Following the removal of the Fmoc protection, the peptideintermediate is first reacted using 2-bromoethyl,2'(Boc-amino)ethylether dissolved in DMF for 1 hour or until the ninhydrin test isnegative to link the carbon atom to the side-chain amino group by theremoval of the halogen to form: Q--NH--(CH₂)₂ --O--(CH₂)₂ --NH(Boc).This compound is then reacted as generally described in Example I usingPCI to form the cyanoguanidino moiety with the side chain secondaryamino group of the residue in the 6-position. Next the Boc protectiongroup is removed, and the primary amino group reacts with the -OPh groupto give the compound: ##STR48## Following cleavage and HPLC purificationas previously described, the GnRH antagonist (Peptide No. 149) istested. The peptide is considered to be effective to prevent ovulationof female mammals at low dosages.

EXAMPLE XVI I

(Boc)Dpr is reacted with PCI as in Example I to produce ##STR49## whichis then reacted with hydrazine dissolved in DMF for 1 to 2 days at roomtemperature, washed with DMF and then repeated to replace the --OPhgroup with concomminent formation of the heterocycle: ##STR50## which isparticularly useful as a substitute for His in peptide syntheses.

EXAMPLE XVIII Preparation of D and L N.sup.α -Boc-4-(5'-amino-1H-1',2',4'-triazollyl))-Aminophenylalanines from D and L phenylalanine

Preparation of the amino acid 3-(4-nitrophenyl)alanine has frequentlyutilized various means of aromatic ring nitration on phenylalanine. Thenitration procedure most frequently used employs a solution of nitricacid dissolved in sulfuric acid and is commonly referred to as a "mixedacid". Contrary to the high yields often reported, it was later foundthat actual nitration occurs in all possible locations of the aromaticportion of the amino acid (ortho, meta and para), and modification ofthe reaction conditions only brought about minor changes in the isomericratios. Repeated crystallizations from boiling water could remove theundesired o- and m-isomers, but the yields were only about 25% of thetheoretical. Although other nitration compositions consistingessentially of nitric acid and acetic anhydride or nitrate salts invarious acids have been used as well, there appeared to be no advantageto these variations of nitration because yields were modest and theisomeric distributions were not reported.

Of the various methods cited in the literature, one factor of thereaction remains constant, i.e. the ratio of the nitrating agent to thequantity of Phe used as a reactant. The value is generally 1.1 to 1.3equivalents of appropriate nitrate to avoid excess nitration. Excesssulfuric acid is generally added to the reaction to help in theformation of the nitration agent (NO⁺²) because nitrations are thoughtto be most rapid in systems which are above 90% sulfuric acid.

It has now been found that the use of larger amounts of sulfuric acidmay lead to a yellow amino acid during the isolation; however, bygreatly increasing the amount of nitric acid so as to provide at leastabout 2 moles (equivalents) of HNO₃ per mole of amino acid, andpreferably between about 2 and about 3.5 equivalents, a cleaner productis produced. Preferably, sulfuric acid is provided in an amount ofbetween about 2 and about 3 equivalents per equivalent of nitric acid.Moreover, duration of action in conjugation gave higher yields. Afterrecrystallization from water, the 3-(4-nitrophenyl)alanine was checkedfor potential contamination of unwanted meta- or ortho-substitutedisomers by use of CZE, and under conditions known to elute the otherisomers, only the para-substituted isomer was seen. The α-amino group isthen protected with an aliphatic urethane protecting group, preferablyBoc.

It is then shown that both D- and L-Boc-4-Aph(atz) amino acids can bereadily prepared from the corresponding Boc-4-Aph amino acids byreaction of the para-amino function with diphenylcyanocarbonimidate,followed by treatment with hydrazine hydrate. This two-step reaction isessentially a one-pot procedure, and although a concentration/solventexchange is carried out between the two steps, such is unnecessary ifthe reaction is done in an alcoholic solvent, such as isopropanol. Thelimited solubility of Boc-4-Aph(atz) in dichloromethane is overcome byusing N-methylpyrrolidine (NMP) as a co-solvent.

The experimental conditions set forth hereinafter are illustrative ofthe preparation of these amino acids.

4-Nitrophenyl-D-Alanine: Into a 600 mL beaker equipped with a stirringbar and maintained in an ice bath, cold, concentrated (18 molar)sulfuric acid (200 mL) was added slowly to 150 mL of cold, concentrated(16 molar) nitric acid (about 2.4 mole). After cooling to 3° C.,D-phenylalanine (180 g, 1.09 mole) was added in 5-10 gram portions over1 hour to ensure that the temperature stayed about 5° C. The reactionwas stirred at ice bath temperature (2°-5° C.) for 4 hours. The reactionwas then quenched by pouring onto 3 kg of ice, followed by cautiousneutralization of the reaction with concentrated ammonium hydroxide. AtpH 5-6, the solution became turbid, and 4-nitrophenyl-D-alanineprecipitated. The precipitate was collected by filtration and washedwith ice water (1L). This amino acid was then twice recrystallized from700 mL of hot water. The recrystallized product was collected and driedunder high vacuum to give 157 grams of 4-nitrophenyl-D-alanine (0.75mole; 68%). α!_(D) =-6.9° (c=0.86 in 1N HCl); mp=238°-244° C. (lit.238°-240° C.).

4-Nitrophenyl-L-Alanine: The reaction described above for4-nitrophenyl-D-alanine was repeated for the L isomer. About 125 gramsof L-Phe was nitrated as described above and resulted in about 124 gramsof 4-nitrophenyl-L-alanine (about 77% yield). α!_(D) =+6.8° (c=1.0 in 1NHCl).

N.sup.α -Boc-4-Nitrophenyl-D-Alanine: 4-Nitrophenyl-D-alanine (157grams, 0.75 mole) was dissolved in 500 mL of 40% aqueous tert-butylalcohol, and the pH was adjusted to 9.9 with 2N NaOH.Di-tert-butyldicarbonate (198 g, 0.9 mole) was added over 35 minutes tothe stirred reaction, and the mixture was then allowed to stir atambient temperature for 2 hours. The pH was maintained at about 9.8 overthe course of the reaction. The reaction product was then firstextracted with petroleum ether (2×800 mL) and diethyl ether (1×500 mL),followed by cautious acidification of the aqueous phase to pH 2 withNaHSO₄. The aqueous layer was extracted with ethyl acetate; this wasthen dried over MgSO₄ and concentrated under vacuum to yield a viscousoil that solidified upon trituration with petroleum ether. The Boc-aminoacid was collected by filtration, washed with petroleum ether and driedto give 210 g of N.sup.α -Boc-4-nitrophenyl-D-alanine (0.68 mole, 90%).α!_(D) =-7.0° (c=0.9 in MeOH); mp=100°-104° C. (lit. 108°-110° C.).

N.sup.α -Boc-4-Nitrophenyl-L-Alanine: The reaction described above forN.sup.α -Boc-4-Nitrophenyl-D-alanine was repeated for the L-isomer.About 123 grams of 4-nitrophenyl-L-alanine was reacted as described andresulted in about 155 grams of N.sup.α -Boc-4-Nitrophenyl-L-alanine(about 86% yield). α!_(D) =+7.1° (c=1.0 in MeOH).

N.sup.α -Boc-4-Aminophenyl-D-Alanine: N.sup.α-Boc-4-Nitrophenyl-D-alanine (209 grams, 0.68 mole) was dissolved in 500mL of ethyl alcohol and acidified with acetic acid (10 mL). To this wasadded 0.65 g of 10% Pd/carbon, and the mixture was hydrogenated at aboutroom temperature using H₂ at about 40 psi until the uptake of hydrogenhad ceased (6 hours). The catalyst was removed by filtration, and thereaction products were concentrated under vacuum to yield a viscous oilthat solidified upon trituration with petroleum ether. The amino acidwas collected by filtration, washed with petroleum ether and dried togive 184 g of N.sup.α -Boc-4-aminophenyl-D-alanine (0.659 mole, 97%)having α!_(D) =-40.2° (c=1.0 in EtOAc) and -23.4° (c=1.0 in MeOH);mp=128°-133° C.

N.sup.α -Boc-4-Aminophenyl-L-Alanine: The reaction described above forN.sup.α -Boc-4-aminophenyl-D-alanine was repeated for the L-isomer.About 153 grams of N.sup.α -Boc-4-nitrophenyl-L-alanine was reacted asdescribed above and resulted in about 134 grams of N.sup.α-Boc-4-aminophenyl-L-alanine (about 96% yield) α!_(D) =+23.6° (c=1.0 inMeOH).

N.sup.α-Boc-4-(5'-(3'-amino-1H-1',2',4'-triazolyl))-Aminophenyl-L-AlanineBoc-L-4Aph(atz)!: To 9 grams of Boc-L-4-aminophenylalanine (32 mmol) ina 250 mL round bottom flask equipped with a mechanical stirrer was added100 mL of dichloromethane and enough N-methylpyrrolidone until all thesolid had dissolved (10 mL). At such time, 8.7 grams (36.6 mmol) ofdiphenylcyanocarbonimidate was added, and the reaction mixture wasstirred at ambient temperature for about 17 hours. The reaction mixturewas then concentrated under vacuum, and the oil obtained was dissolvedin 125 mL of methanol. 15 mL of hydrazine hydrate was added, and thereaction mixture was stirred at ambient temperature for 24 hours. Thisreaction mixture was then concentrated under vacuum to approximately 50mL, and the oil obtained was diluted into 350 mL of cold water. Dilutesulfuric acid was added until the pH was about 7, and the aqueous layerwas extracted with ether (1×150 mL) to remove the phenol formed in thereaction. The pH of the aqueous layer was then adjusted to 2-3, at whichtime a precipitate formed. The reaction product was then extracted withhot ethyl acetate (3×250 mL), and the organic layer dried over MgSO₄.The organic layer was then concentrated to give an oil which, uponwashing with ether, solidified. The powder obtained was filtered, washedwith petroleum ether, and then dried to give 9.2 g of Boc-L-Aph(atz) asa tan powder (25.3 mmol, 79% yield) which is also referred to asBoc-Aph(3-amino, 1,2,4 triazole). α!_(D) =-16.4° (c=1.0 in MeOH).

N.sup.α-Boc-4-(5'-(3'-amino-1H-1',2',4'-triazolyl))-aminophenyl-D-alanineBoc-D-4Aph(atz)!: The reaction described above for the Boc-L-Aph(atz)was repeated for the D-isomer. 10.1 grams of Boc-D-Aph was treated with10.8 grams of diphenylcyanocarbonimidate followed by 20 mL of hydrazinehydrate; it eventually resulted in 7.8 grams of Boc-D-Aph(atz) (61%yield), having an α!_(D) =-15.6° (c=1.0 in MeOH).

EXAMPLE XIX

The decapeptide Ac-β-D-2NAL¹, (4Cl)D-Phe², D-3PAL³, Aph(atz)⁵,D-Aph(atz)⁶, ILys⁸, D-Ala¹⁰ !-GnRH is synthesized by solid-phasesynthesis. This peptide has the following formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Aph(3-amino, 1,2,4triazole)-D-Aph(3-amino, 1,2,4triazole)-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂.

MBHA resin (0.76 mM/g) is used, and Boc-protected D-Ala is coupled tothe resin over a 2-hour period in CH₂ Cl₂ using about a two-fold excessof Boc derivative and DCC as an activating reagent. The D-Ala residueattaches to the MBHA residue by an amide bond.

Beginning with about 0.9 grams of resin and using an automated machine,coupling of each amino acid residue, washing, deblocking and thecoupling of the next amino acid residue is carried out in accordancewith the following schedule:

    ______________________________________                                                                        MIX                                                                           TIMES                                         STEP REAGENTS AND OPERATIONS    MIN.                                          ______________________________________                                        1    CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                                  3                                             2    Methanol (MeOH) wash-30 ml. (2 times)                                                                    3                                             3    CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                             4    50 percent TFA plus 5 percent1,2-ethanedithiol                                                           10                                                 or m-cresol in CH.sub.2 Cl.sub.2 -70 ml. (2 times)                       5    Isopropyl alcohol wash-80 ml. (2 times)                                                                  3                                             6    TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                            5                                             7    MeOH wash-40 ml. (2 times) 2                                             8    TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                            5                                             9    CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                                  3                                             10   Boc-amino acid (3.5 mmoles) in 30 ml. of either                                                           30-300                                            m-methylpyrrolidine (NMP): CH.sub.2 Cl.sub.2 or NMP alone,                    depending upon the solubility of the particular                               protected amino acid, plus DIC or DCC (3.5                                    mmoles) in CH.sub.2 Cl.sub.2                                             11   MeOH wash-40 ml. (2 times) 3                                             12   Triethylamine (TEA) 12.5 percent in CH.sub.2 Cl.sub.2 -70                                                3l.                                           13   MeOH wash-30 ml. (2 times) 3                                             14   DCM wash-80 ml. (2 times)  3                                             ______________________________________                                    

The above schedule is used for coupling of each of the amino acids ofthe peptide of the invention after the first amino acid has beenattached. N.sup.α Boc protection is used for each of the remaining aminoacids throughout the synthesis. N.sup.α Boc-β-D-2NAL is prepared by amethod known in the art, e.g. as described in detail in U.S. Pat. No.4,234,571, issued Nov. 18, 1980; it is also commercially available fromSyntheTech, Oregon, U.S.A. Bzl(benzyl ether) is used as a side chainprotecting group for the hydroxyl group of Ser. Boc-Lys(Ipr,Z) is usedfor the 8-position. The side chain groups of 4Aph(atz) in the 5-positionand D-4Aph(atz) in the 6-position need not be protected. Afterdeblocking the α-amino group at the N-terminus using trifluoroaceticacid (TFA), acetylation is achieved using a large excess of aceticanhydride in dichloromethane.

Following completion of the assembly of the peptide and acetylation ofthe N-terminus, about 1.67 grams of the following intermediate arepresent:Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Aph(atz)-D-Aph(atz)-Leu-Lys(Ipr,Z)-Pro-D-Ala-NH-MBHA resin support!.

The peptidoresin is dried, and then cleavage of the peptide from theresin and deprotection of the Ser and the Lys side chains is carried outat 0° C. with HF. Anisole is added as a scavenger prior to HF treatment.After the removal of HF under vacuum, the resin is washed twice with 100ml. of ethyl ether. The cleaved peptide is extracted from the resin withequal parts of CH₃ CN and H₂ O, repeating the process and using 150 ml.each time. The extracts are pooled and lyophilized, and they provideabout 650 mg of a crude peptide powder.

Purification of the peptide is then effected by preparative highperformance liquid chromatography (HPLC), as known in the art andspecifically set forth in J. Rivier, et al. J. Chromatography, 288,303-328 (1984). The first preparative RP-HPLC separation uses a TEAP(triethylammonium phosphate) buffer system. This separation is repeatedusing the same buffer system with a slightly different gradient, and thefinal separation is carried out using a 0.1% TFA (trifluoroacetic acid)gradient, all as described in detail in the J. Chromatography article.About 80.7 milligrams of the decapeptide are obtained.

The peptide is judged to be homogeneous using capillary zoneelectrophoresis (CZE), as well as by using reversed-phase highperformance liquid chromatography and an aqueous triethylammoniumphosphate buffer plus acetonitrile. The purity is estimated to be about97%, and the peptide coelutes with peptide 103A as prepared using thesynthesis described in detail in Example VIII, showing that the samecompound is formed by this synthesis. Amino acid analysis of theresultant, purified peptide is consistent with the formula for theprepared structure, showing substantially integer-values for each aminoacid in the chain; mass spectral analysis is also consistent. Theoptical rotation is measured on a photoelectric polarimeter and found tobe the same as that measured for Peptide No. 103A.

The peptides of the invention are often administered in the form ofpharmaceutically acceptable, nontoxic salts, such as acid additionsalts, or of metal complexes, e.g., with zinc, barium, calcium,magnesium, aluminum or the like (which are considered as addition saltsfor purposes of this application), or of combinations of the two.Illustrative of such acid addition salts are hydrochloride,hydrobromide, sulphate, phosphate, nitrate, oxalate, fumarate,gluconate, tannate, maleate, acetate, citrate, benzoate, succinate,alginate, malate, ascorbate, tartrate and the like. For example, anaqueous solution of the peptide can be repeatedly treated with 1N aceticacid and then lyophilized to yield the acetic acid salt thereof. If theactive ingredient is to be administered in tablet form, the tablet maycontain a pharmaceutically-acceptable diluent which includes a binder,such as tragacanth, corn starch or gelatin; a disintegrating agent, suchas alginic acid; and a lubricant, such as magnesium stearate. Ifadministration in liquid form is desired, sweetening and/or flavoringmay be used as part of the pharmaceutically-acceptable diluent, andintravenous administration in isotonic saline, phosphate buffersolutions or the like may be effected.

The pharmaceutical compositions will usually contain the peptide inconjunction with a conventional, pharmaceutically-acceptable carrier.Usually, the dosage will be from about 10 micrograms to about 0.5milligram of the peptide per kilogram of the body weight of the hostwhen given intravenously, intramuscularly or subcutaneously; althoughoral dosages will be higher, it is anticipated that the nature of thesecompounds will permit effective oral administration. Overall, treatmentof subjects with these peptides is generally carried out in the samemanner as the clinical treatment using other antagonists or agonists ofGnRH using a suitable carrier in which the peptide is soluble.

It may also be desirable to deliver the GnRH analog over prolongedperiods of time, for example, for periods of one week to one year from asingle administration, and slow release, depot or implant dosage formsmay be utilized. For example, a dosage form may contain apharmaceutically acceptable non-toxic salt of the compound which has alow degree of solubility in body fluids, for example, an acid additionsalt with a polybasic acid; a salt with a polyvalent metal cation; orcombination of the two salts. A relatively insoluble salt may also beformulated in a gel, for example, an aluminum stearate gel. A suitable,slow-release depot formulation for injection may also contain the GnRHanalog or a salt thereof dispersed or encapsulated in a slow degrading,non-toxic or non-antigenic polymer such as a polylacticacid/poly-glycolic acid polymer, for example, as described in U.S. Pat.No. 3,773,919. These compounds may also be formulated into silasticimplants or as pellets in cholesterol.

These peptides can be administered to mammals intravenously,subcutaneously, intramuscularly, orally, percutaneously, e.g.intranasally or intravaginally to achieve fertility inhibition and/orcontrol and also in applications calling for reversible suppression ofgonadal activity, such as for the management of precocious puberty orduring radiation- or chemotherapy. They are also useful for treatment ofsteroid-dependent tumors. Effective dosages will vary with the form ofadministration and the particular species of mammal being treated. Anexample of one typical dosage form is a bacteriostatic water solutioncontaining the peptide which solution is administered parenterally toprovide a dose in the range of about 0.1 to 2.5 mg/kg of body weight perday. Oral administration of the peptide may be given in either solidform or liquid form.

Although the invention has been described with regard to its preferredembodiments, it should be understood that changes and modifications aswould be obvious to one having the ordinary skill in this art may bemade without departing from the scope of the invention which is setforth in the claims which are appended hereto. For example, othersubstitutions on the peptide chain known in the art which do notsignificantly detract from the effectiveness of the peptides may beemployed in the peptides of the invention. D-2PAL and D-4PAL areconsidered to be equivalents of D-3PAL. Other equivalent acylatinggroups can be used instead of acetyl at the N-terminus. The 6-positionsubstitutes set forth in Table VI are considered to be equivalents knownin the prior art and can be included in the peptides of the invention.Substituted Phe, such as (4F)Phe, can be used instead of Phe in the7-position. Both butyl Lys and diethyl Lys are considered to beequivalents of ILys; however, ILys is preferred when neither U* or Argis in the 8-position. Homoarginine and norarginine, i.e. H₂ NC(═NH)NHCH₂CH₂ CH(NH₂)COOH, are considered the equivalent of Arg in this position.Other hydrophobic amino acid residues can also be employed in the1-position, preferably in D-isomer form, and are considered equivalentsof those specified. Moreover, the analogs can be administered in theform of their pharmaceutically or vetinarially acceptable, nontoxicsalts, as indicated hereinbefore, which are considered equivalents. Inthe amino acids, instead of making the modifications upon Phe eitherhomoPhe (Hph) or homohomoPhe (Hhp) can be used to create unnatural aminoacids having similar properties in either the D- or L-isomer forms.

Particular features of the invention are emphasized in the claims whichfollow.

What is claimed is:
 1. An amino acid having the formula: ##STR51##wherein J is 1, 2 or 3, Y is N--CN, CH--NO₂ or N--CONH₂, and R₂ is alkyl(C₁ to C₆), modified alkyl (C₁ to C₅ with terminal substitution by NH₂,OH, Cl, Br, F or F₃), alkenyl (C₂ to C₄), alkynyl (C₂ to C₄), benzyl,tolyl, p-amino-benzyl, p-chloro-benzyl, or methylpyridyl.
 2. An aminoacid having the formula: ##STR52## wherein j is 1, 2 or 3, Y is N--CN,CH--NO₂ or N--CONH₂, and R₂ is alkyl (C₁ to C₆), modified alkyl (C₁ toC₅ with terminal substitution by NH₂, OH, Cl, Br, F or F₃), alkenyl (C₂to C₄), alkynyl (C₂ to C₄), benzyl, tolyl, p-amino-benzyl,p-chloro-benzyl, or methylpyridyl.
 3. An amino acid according to claim 1or 2 wherein j is
 1. 4. An amino acid according to claim 1 or 2 whereinY is N--CN.
 5. An amino acid according to claim 5 wherein X is NH and R₂is methylpyridyl.
 6. An amino acid according to claim 5 wherein X is NHand R₂ is butyl.
 7. A method of synthesizing a peptide by chainelongation from the C-terminus thereof, which method comprisessubjectingan α-amino acid having the formula: ##STR53## to nitration conditions byforming a reaction mixture which includes said amino acid, concentratedsulfuric acid and concentrated nitric acid having at least about 2 molesof HNO₃ for each mole of α-amino acid and maintaining said reactionmixture at a temperature of about 5° C. or below to cause said nitrationto occur predominantly at the 4-position, reacting said 4NO₂-substituted product with an appropriate reagent to add anamino-protecting group to said α-amino acid, treating said protectedamino acid to hydrogenate said substituted nitro moiety and transform itto an amino moiety, dissolving said hydrogenated α-amino acid in asuitable solvent and reacting it with diphenylcyanocarbonimidate to forma cyanoguanidino intermediate, thereafter reacting said cyanoguanidinointermediate with hydrazine to create a 3-amino, 1,2,4 triazole moietyattached to the 4-position of the phenyl ring of the side chain of saidα-amino acid, forming a peptide intermediate with a protected α-aminogroup at its N-terminus, and deprotecting the α-amino group of saidpeptide intermediate and reacting same to form an amide bond with saidα-amino acid having said triazole side chain.
 8. A method ofsynthesizing a peptide by chain elongation from the C-terminus thereof,which method comprisesforming a peptide intermediate having a protectedα-amino group at its N-terminus, deprotecting the α-amino group of saidpeptide intermediate and reacting said deprotected peptide intermediateto form an amide bond with an α-amino acid according to claim 1, havingthe formula: ##STR54## wherein the α-amino group is protected.
 9. Amethod of synthesizing a peptide by chain elongation from the C-terminusthereof, which method comprisesforming a peptide intermediate whichincludes the amino acid sequence Leu-Lys(Ipr)-Pro-D-Ala, causing a firstreaction between the α-amino group of Leu at the N-terminus of saidpeptide intermediate with the α-carboxyl group of Boc-D-Aph(3-amino,1,2,4 triazole) to elongate said peptide intermediate, removing said Bocprotecting group from said elongated peptide intermediate, carrying outa second reaction with the e-carboxyl group of Boc-L-Aph(3-amino, 1,2,4triazole) to further elongate said elongated peptide intermediate,removing said Boc protecting group and sequentially adding Boc-Ser,Boc-D-3PAL, Boc(4Cl)D-Phe and Boc-β-D-2Nal to create a decapeptideintermediate, removing the Boc protecting group from the N-terminus ofsaid decapeptide intermediate and acetylating said deprotectedN-terminus, and removing any remaining protecting groups to provide thedecapeptide Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Aph(3-amino, 1,2,4triazole)-D-Aph(3-amino, 1,2,4triazole)-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂.
 10. A method according toclaim 9 wherein Boc-D-4Aph(3-amino, 1,2,4 triazole) is used in saidfirst reaction and Boc-L-4Aph(3-amino, 1,2,4 triazole) is used in saidsecond reaction.